CA2501872A1 - Screening systems and methods for identifying modulators of xenobiotic metabolism - Google Patents

Abstract

The present invention provides mice having reduced CAR receptor activity and mice expressing a human CAR receptor. These mice are useful in screening methods to identify CAR ligands, including compounds that modulate CAR
receptor activity, compounds likely to have CAR-mediated toxicity, and analogs of these compounds with less potential toxicity.

Description

SCREENING SYSTEMS AND METHODS FOR IDENTIFYING
MODULATORS OF XENOBIOTIC METABOLISM
Cross-Reference to Related Applications This application is a continuation-in-part of International Application PCT/LTSOl/29672, filed September 21, 2001' and published in English, and a continuation-in-part of U.S. utility application 10/219,590, filed August 15, 2002, both of which claim priority to U.S. utility application 091666,250, filed September 21, 2000.
Statement as to Federally Sponsored Research This invention was made with government support under NIH grant N1DDK RO 1 DK46546. The government therefore has certain rights in the invention.
Background of the Invention In general, the present invention is directed to compositions, including transgenic animals, and methods fox identifying modulators of metabolism of any of a wide range of foreign compounds, collectively termed xenobiotics.
A number of cytochrome P450 (CYP) enzymes able to metabolize diverse substrates serve as a primary defense against potentially deleterious effects of xenobiotic compounds. Induction of the expression of individual CYP genes in response to particular xenobiotics is a central component of this metabolic mechanism.
One,of the best characterized of these responses is the induction of specific CYP genes by a diverse group of agents known as "phenobarbital-like" inducers. Exposure of animals to any of a chemically diverse series of compounds exemplified by phenobarbital (PB) results in a potent activation of expression of a specific subset of CYP enzymes and other proteins associated with xenobiotic metabolism. In the mouse, these PB-like inducers increase expression of CYP2B 10 and several other genes. The pesticide contaminant 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, referred to as TCPOBOP, is generally considered to be the most potent of this group of inducers.

Xenobiotics, such as therapeutic drugs, insecticides, polycyclic hydrocarbons, and some natural products, are often metabolized via oxidation reactions catalyzed by CYP
enzymes. These reactions add hydrophilic groups to xenobiotics, allowing the body to rid itself of these noxious or simply insoluble materials. For example, oxidation of polycyclic aromatics produces epoxides, which are very reactive electrophilic groups.
Usually these epoxides are rapidly hydrolyzed into hydroxyl groups which are then coupled to other groups, producing compounds water-soluble enough to be excreted.
Unfortunately, the intermediate epoxides may also be released into the cell as highly reactive electrophiles, possibly reacting with negatively charged groups in DNA.and causing changes in the DNA sequence. Reactive oxygen species generated during metabolism of cocaine by CYP enzymes in humans has been associated with mutagenesis and chromosome breakage.
CYP-mediated metabolism may also result in other undesired effects, such as the rapid degradation of a therapeutically active compound, lowering its half-life in vivo.
Alternatively, CYP enzymes may convert a prodrug into an active drug at a faster than desired rate resulting in a toxic concentration of the active drug in vivo.
Additionally, the activation of CYP enzymes by the administration of a therapeutically active compound or exposure to another foreign compound may result in faster metabolism of a second therapeutically active compound, reducing its effectiveness or increasing its toxicity.
Because of the potentially deleterious effects of compounds that activate CYP
enzymes, improved methods are needed to determine which compounds activate CYP-mediated metabolism and, thus, might cause side-effects if administered to humans.
These compounds may thereby be eliminated from drug development or chemically modified to generate related compounds with less ability to activate CYP
enzymes.
Summary of the Invention The present invention provides screening systems and methods that facilitate the identification of compounds that activate or inhibit a Constitutive Activator of Retinoic acid response elements (referred to as "CAR" or "CAR receptor"). Such CAR
receptor-activating compounds are potentially toxic when administered to a mammal alone or in combination with other compounds, and are therefore preferably excluded from candidate drugs or drug development programs. Similarly, compounds that inhibit a CAR
receptor may be administered to a mammal to decrease the CAR-mediated metabolism of a therapeutically active compound, potentially decreasing side-effects and re-establishing the therapeutic half-life of the compound ifZ vivo. Such a decrease of metabolic activity may also be useful to decrease production of toxic products from appropriate precursor compounds. , Accordingly, in a first aspect, the invention features a transgenic mouse expressing a human CAR receptor. Preferably, the human CAR receptor is activated by Phenobarbital. In various embodiments, Phenobarbital increases the rate of human CAR-mediated induction of a CAR target gene or a transgene operably-linked to a CAR
responsive promoter by at least 2, 5, 10, or 20-fold. In certain embodiments, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene increases the rate of human CAR-mediated induction of a CAR target gene or a transgene operably-linked to a CAR
responsive promoter by less than 20, 10, 5, or 2-fold. In other embodiments, an activity of the human CAR receptor (e.g., induction of a CAR target gene) is increased by less than 200, 100, 50, or 25% by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In preferred embodiments, Phenobarbital increases an activity of the human CAR receptor by at least 2, 5, 10, 20, or 40-fold more than the corresponding amount of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene increases the activity of the human CAR
receptor. In other preferred embodiments, administration of acetaminophen to the mouse activates human CAR-mediated induction of a CAR target gene (e.g., CYP1A2, CYP3A11, or GSTPi). Preferably, administration to the mouse of an inhibitor of human CAR (e.g., an inverse agonist) decreases the level of activation of human CAR
(e.g., reduces the induction of CYP1A2, CYP3A11, or GSTPi) by acetaminophen.
In preferred embodiments of the above aspect, the mRNA of the human CAR
receptor includes a bovine growth hormone polyadenylation sequence.
Preferably, the nucleic acid encoding the human CAR receptor is operably linked to an intronic sequence of a rabbit (3-globin nucleic acid, which increases the expression of the human 'CAR
receptor.
In a preferred embodiment, the transgenic mouse expressing a human CAR
receptor does not express a substantially active murine CAR receptor or does not express any murine CAR receptor.
In another aspect, a transgenic mouse expressing human CAR exhibits, induction of a xenobiotic response in the presence of a xenobiotic inducer. In one embodiment, the transgenic mouse of the present invention exhibits an induction or activation of a CAR
target gene, such as CYP2B 10 mRNA in response to PB. In another embodiment, the transgenic mice expressing human CAR does not demonstrate or exhibit an induction or activation of a CAR target gene in response to TCPOBOP.
In yet another aspect, the invention features a mouse having a mutation that reduces CAR receptor activity. Preferably, the activity of a murine CAR
receptor is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% compared the corresponding activity of a naturally-occurring murine CAR receptor. In some preferred embodiments, the mouse does not express a functional murine CAR receptor. In a preferred embodiment, the level of induction of a CAR target gene (e.g., CYP1A2, CYP3A11, or GSTPi) in response to acetaminophen administered to the mouse having a mutation in a CAR receptor is less that 10, 5, or 2-fold times the corresponding level of induction in a CAR null mouse that does not express CAR mRNA or protein.
In a further aspect, the present invention features a transgenic animal deficient of or lacking in a CAR gene. In a preferred embodiment, the transgenic animal of the present invention comprises a reduction or absence of native or naturally-occurring CAR
receptor activity, and is preferably a mouse.
In various embodiments, the transgenic mouse of the present invention exhibits a decrease or absence of induction, expression, or activity of a CAR target gene in response to a xenobiotic inducer. In a preferred embodiment, the transgenic mouse exhibits a decrease or absence of induction, expression, or activity of a CAR target gene. In a particular embodiment, the transgenic mouse of the present invention exhibit a decrease or absence of CYP enzyme induction, activity, or expression in response to a xenobiotic inducer, such as PB or TCPOBOP.
In another embodiment, the trarisgenic mouse of the present invention does not exhibit hyperplasia or hypertrophy of the liver upon treatment with a xenobiotic inducer, including PB or TCPOBOP. Preferably, the transgenic mouse of the present invention does not exhibit hyperplasia or hypertrophy of the liver in response to treatment with either Phenobarbital or TCPOBOP as exhibited in wild-type mice.
In a furCher embodiment, the transgenic mouse exhibits decreased metabolism of the CYP substrate zoxazolamine. In yet a further embodiment, the transgenic mouse exhibits a resistance to acetaminophen toxicity.
The present invention also provides cells derived from the transgenic animals of the present invention, such as cells that are isolated or present in a tissue or organ, and any cell lines or any progeny thereof.
The animals of the present invention may be used to determine whether a compound modulates the activity of a CAR receptor. In addition, methods are provided to determine whether the metabolism of a compound is regulated by modulation of the activity of a CAR receptor.
Accordingly, the invention also features a screening method for determining whether a compound activates a CAR receptor. This method involves administering a compound to a transgenic mouse expressing a human CAR receptor and measuring induction of a CAR target gene. The compound is determined to activate the CAR
receptor if the compound mediates induction of the CAR target gene. In one preferred embodiment, a CAR receptor inverse agonist is also administered to the mouse expressing a human CAR receptor. Preferably, the CAR receptor inverse agonist is clotrimazole.
In another aspect, the invention .features a screening method for deternnining whether a compound inhibits a CAR receptor. This method involves administering the compound to a transgenic mouse expressing a human CAR receptor and measuring expression of a CAR target gene in the presence and absence of the compound.
The compound is determined to inhibit the CAR receptor if the compound decreases the expression of the CAR target gene. In one preferred embodiment, a CAR receptor agonist is also administered to the mouse expressing the human CAR receptor.
Preferably, the CAR receptor agonist is, a functional CAR receptor agonist that is specific for human CAR, and the agonist is administered after the compound is administered to the mouse.
In yet another aspect, the invention features a screening method for determining .
whether a compound modulates the activity of a CAR receptor. This method involves administering the compound to a transgenic mouse expressing a human CAR
receptor and measuring a physiological effect mediated by the administration of the compound.
The compound is determined to modulate the activity of the CAR receptor if the magnitude of the physiological effect iri the mouse expressing the human receptor differs from that in a mouse having a mutation that reduces CAR receptor activity. In preferred embodiments, the physiological effect is assayed by measuring the toxicity or activity mediated by the administration of the compound or by measuring the half-life of the compound. In other preferred embodiments, the toxicity or activity is mediated by a metabolite of the compound. Preferably, the difference between the magnitude of the physiological effect in the mouse expressing the human CAR receptor as compared to a mouse having reduced CAR receptor activity is at least 2, 5, 10, or 20-fold.
In. other preferred embodiments, the magnitude of the physiological effect in a mouse having reduced CAR activity is at least 10, 25, 50, or 75% smaller or larger than the magnitude of the effect in the mouse expressing the human CAR receptor.
In still another aspect, the invention features a screening method for determining whether the metabolism of a compound is regulated by modulation of the activity of a CAR receptor. This method involves administering the compound to a transgenic mouse expressing a human CAR receptor and measuring the rate of metabolism of the compound. The metabolism of the compound is determined to be regulated by modulation of the activity of the CAR receptor if the rate of metabolism is faster in the mouse expressing the human receptor than in a mouse having reduced CAR
receptor activity. Preferably, the rate of metabolism is at least 2, 5, 10, or 20-fold faster in the mouse expressing the human CAR receptor than in the mouse having reduced CAR
receptor activity. In preferred embodiments, the rate of metabolism is determined by measuring the toxicity or activity mediated by the administration of the compound, measuring the half-life of the compound, or measuring the serum level of a liver enzyme.
Preferably, these measurements are performed at more than l, 3, or 5 time points after administration of the compound.
In another aspect, the invention provides a screening method for determining whether the metabolism of a first compound is modulated by a second compound.
This method involves administering the first compound in the presence and absence of the second compound to a transgenic mouse expressing a human CAR receptor. A
physiological effect that is mediated by the administration of the first compound is measured in the presence and absence of the second compound. The second compound is determined to modulate the metabolism of the first compound if the second compound effects a change in the physiological effect mediated by the administration of the first compound. In preferred embodiments, the physiological effect is assayed by measuring the toxicity or activity mediated by the administration of the first compound or measuring the half-life of the first compound. In various preferred embodiments, the toxicity or activity is mediated by a metabolite of the first compound. In still another preferred embodiment, the physiological effect is assayed by measuring the half-life of the first compound in the presence and absence of the second compound. The second compound is determined to activate the metabolism of the first compound if the second compound decreases the half-life, or the second compound is determined to inhibit the metabolism of the first compound if the second compound increases the half-life.
Similar methods for determining whether a compound modulates the activity of a CAR receptor or the metabolism of another compound may also be performed using a mouse having a mutation that reduces CAR receptor activity. For example, the invention features a screening method for determining whether a compound activates a CAR
receptor. This method involves administering a compound to a mouse having a mutation that reduces CAR receptor activity and measuring induction of a CAR target gene. The compound is determined to activate the CAR receptor if the induction is smaller in the mouse having reduced CAR receptor activity than in a mouse having wild-type CAR
receptor activity. In a preferred embodiment, a CAR receptor inverse agonist to is also administered to the mouse having reduced CAR receptor activity. Preferably, the inverse agonist is androstanol.
In another aspect, the invention features a screening method for determining whether a compound inhibits a CAR receptor. This method involves administering the compound to a mouse having a mutation that reduces CAR receptor activity and measuring expression of a CAR target gene in the presence and absence of the compound. The compound is determined to inhibit the CAR receptor if the decrease in expression effected by the compound is smaller in the mouse having reduced CAR

receptor activity than in a mouse having wild-type CAR receptor activity. In one preferred embodiment, a CAR receptor agonist is also administered to the mouse having reduced CAR receptor activity. Preferably, the CAR receptor agonist is TCPOBOP, and the TCPOBOP is administered after the compound.
In still another aspect, the invention features a screening method for deteimining whether a compound modulates the activity of a CAR receptor. This method involves administering the compound to a mouse having a mutation that reduces CAR
receptor activity and measuring a physiological effect mediated by the administration of the compound. The compound is determined to modulate the activity of the CAR
receptor if the magnitude of the physiological effect in the mouse having reduced CAR
receptor activity differs from that in a mouse having wild-type CAR receptor activity.
Preferably, the difference between the magnitude of the physiological effect in the mouse having reduced CAR receptor as compared to a mouse having wild-type CAR receptor activity is at least 2, 5, 10, or 20-fold. In other preferred embodiments, the magnitude of the physiological effect in the mouse having reduced CAR activity is at least 10, 25, 50, or 75% smaller or larger than the magnitude of the effect in a mouse having wild-type CAR
receptor activity. In yet other preferred embodiments, the physiological effect is assayed by measuring the toxicity or activity mediated by the administration of the compound or measuring the half-life of the compound. In another preferred embodiment, the toxicity or activity is mediated by a metabolite of the compound.
In still another aspect, the invention provides a screening method for determining whether the metabolism of a compound is regulated by modulation of the activity of a CAR receptor. This method involves administering the compound to a mouse having a mutation that reduces CAR receptor activity and measuring the rate of metabolism of the compound. The metabolism of the compound is determined to be regulated by modulation of the activity of the CAR receptor if the rate of metabolism is slower in the mouse having reduced CAR receptor activity than in a mouse having wild-type CAR
receptor activity. Preferably, the rate of metabolism is at least 2, 5, 10, or 20-fold slower in the mouse having reduced CAR receptor activity than in a mouse having wild-type CAR receptor activity.
In preferred embodiments, the rate of metabolism is determined by measuring the toxicity or activity mediated by the administration of the compound, measuring the half-life of the compound, or measuring the serum level of a liver enzyme.
Preferably, these measurements are performed at more than 1, 3, or 5 time points after administration of the compound.
In yet another aspect, the invention.features a screening method for determining whether the metabolism of a first compound is modulated by a second compound.
This method involves administering the first compound in the presence and absence of the second compound to a mouse having a mutation that reduces CAR receptor activity. A
physiological effect that is mediated by the administration of the first compound is measured in the presence and absence of the second compound. The second compound is determined to modulate the metabolism of the first compound if the change effected by the second compound in the physiological effect mediated by the administration of the first compound is smaller in the mouse.having reduced CAR receptor activity than in a mouse having wild-type CAR receptor activity. In preferred embodiments, the physiological effect is assayed by measuring the toxicity or activity mediated by the administration of the first compound or measuring the half-life of the first compound. In various preferred embodiments, the toxicity or activity is mediated by a metabolite of the first compound. In another preferred embodiment, the physiological effect is assayed by measuring the half-life of the first compound in the presence and absence of the second compound. The second compound is determined to activate the metabolism of the first compound if the decrease in the half-life effected by the second compound is smaller in the mouse having reduced CAR receptor activity than in a mouse having wild-type CAR
receptor activity, or the second compound is determined to inhibit the metabolism of the first compound if the increase in the half-life effected by the second compound is smaller in the mouse having reduced CAR receptor activity than in a mouse having wild-type CAR receptor activity.
. The above screening methods may be carried out using transgenic mouse expressing a human CAR receptor, for example, any such transgenic mouse described herein.
The invention also features methods for preventing, stabilizing, or treating toxicity associated with CAR-mediated.metabolisrn of a xenobiotic by administering an inhibitor of CAR receptor expression and/or activity to a mammal (e.g., a human).
Accordingly, in one such aspect, the invention features a method for preventing, stabilizing, or treating an adverse effect or symptom associated with one or more hepatotoxic agents (e.g., acetaminophen and/or phenobarbital) in a mammal (e.g., a human) by administering to a mammal (for example, a human) one ox more compounds that inhibit the expression and/or activity of a CAR receptor in the mammal.
In preferred embodiments of various aspects of the invention, the mouse having a mutation that reduces CAR receptor activity is a transgenic animal.
Preferably, the mutation that reduces CAR receptor activity substantially eliminates CAR
receptor ~ activity. In yet other preferred embodiments, the mouse having a mutation that reduces CAR receptor activity and the mouse having wild-type CAR receptor activity have the same genotype except for a mutation in the CAR receptor gene, promoter, or regulatory sequence. In still other preferred embodiments, the mouse having wild-type CAR
receptor activity is a transgenic mouse expressing a human CAR receptor.
Preferably, the mouse expressing a human CAR receptor does not express a substantially active murine CAR receptor or does not express any murine CAR receptor.
In other preferred embodiments, at least one of the compounds tested in the screening methods of the invention is a member of a library of as few as 2 or compounds to as many as 10, 20, 50, or more compounds, all of which are simultaneously administered to the mouse. Preferred routes of administration of the compounds include oral, intramuscular, intravenous, parenteral, intraarticular, intraperitoneal, subcutaneous, or any other suitable route. Preferably, a compound that activates a CAR receptor or a compound whose metabolism is regulated by modulation of the activity of a CAR receptor is eliminated from drug development. If a first compound activates the metabolism of the second compound, then the first compound, the second compound, or both compounds are preferably eliminated from drug development. It is also contemplated that~other animal models, such as a rat or other rodent having reduced CAR receptor activity or expressing a human CAR
receptor, could be used in any of the various aspects of the invention.
The present invention provides a number of advantages. For example, the methods of the present invention may be used to facilitate the identification of. analogs of a compound that have reduced or undetectable ability to activate a CAR
receptor, and thus are expected to have fewer side-effects or a longer half-life in vivo. In addition, because murine and human CAR receptors have somewhat different substrate specificities, the use of transgenic mice expressing a human CAR receptor in the methods of the present invention may more accurately predict the modulation of CAR
receptor toxicity or half-life of a compound when administered to humans. Moreover, the present assays may be easily and rapidly performed.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.
Definitions The following terms have the meanings ascribed to them below unless specified otherwise.
As used herein, "CAR receptor activity" refers to CAR-mediated induction of a gene, denoted a "CAR target gene," or a transgene operably-linked to a CAR
responsive promoter.
The term "mutation" refers to an alteration in a nucleic acid sequence such that the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid alteration as compared to the naturally-occurring sequence. The mutation may, without limitation, be an insertion, deletion, frameshift mutation, or missense mutation.
Alternatively, the mutation may alter the sequence of a CAR receptor promoter, transcriptional regulatory sequence, or translational regulatory sequence such that a smaller amount of CAR mRNA or protein is expressed. Preferably, the mutation results in at least a 25, 35, 50, 70, 80, 90, 95, 99, or 100% reduction in the activity of the encoded CAR receptor compared to the activity of a naturally-occurring CAR
receptor.
In another preferred embodiment, the level of induction of a CAR target gene in response to a xenobiotic administered to a mouse having a mutation in a CAR receptor is less that 10, 5, or 2-fold times the corresponding.level of induction in a CAR null mouse that does not express CAR mRNA or protein.
The term "transgenic" refers to any cell or organism which includes a DNA
sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organism is generally a transgenic non-human mammal, preferably, a rodent such as a mouse.
The phrase "substantially eliminates CAR receptor activity" means the reduction of CAR receptor activity by 25, 35, 50, 70, 80, 90, 95, 99, or 100% compared to the activity of a naturally-occurring CAR receptor. In another preferred embodiment, the level of residual CAR receptor activity is no greater than 10, 5, or 2 times the corresponding level of CAR receptor activity in a CAR null mouse that does not express CAR mRNA or protein.
The phrase "a substantially active murine CAR receptor" means having at least 30, 60, 80, 90, 95, or 100% of the CAR receptor activity of the naturally-occurring murine CAR receptor encoded by GenBank Accession No. 2267575 in a normal murine host (Choi et al., J. Biol. Chem. 272:235.65-23571, 1997) (SEQ DJ NO: 1).
The phrase "a human CAR receptor" means a protein that has an amino acid sequence at least 75, 80, 90, 95, 99, or 100% identical to the amino acid sequence of the naturally-occurring human CAR receptor, encoded by GenBank Accession No.

(Baes et al., Mol. Cell. Bio. 14:1544-1551, 1994) (SEQ II7 NO: 2), and that has at least 50, 75, 80, 90, 95, or 100% of the CAR receptor activity of a naturally-occurring human CAR receptor assayed under identical conditions. It is also contemplated that the expressed human CAR receptor may be a fragment having an amino acid sequence at least 75, 80, 90, 95, 99, or 100% identical to the corresponding region of a naturally-occurring human CAR receptor and having at least 60, 80, 90, 95, or 100% of the CAR
receptor activity of a naturally-occurring.human CAR receptor. In addition, a human CAR receptor is inhibited by clotrimazole, an inverse agonist of human, but not murine, CAR (Moore et al., J Biol Chem. 275:15122-15127, 2000).
As used herein "naturally-occurring murine or human CAR receptor" refers to a protein with an amino acid sequence identical to that of a murine or human CAR
receptor found in nature, such as the murine CAR receptor encoded by GenBank Accession No.
2267575 or the human CAR receptor encoded by GenBank Accession No. 458541.
The phrase "activation of a CAR receptor" means an increase in the rate of the CAR-mediated induction of a CAR target gene, or a transgene operably-linked to a CAR
responsive promoter. Preferably, the increased induction of the CAR target gene or transgene in a mouse results in a 2, 5, 10, or 20-fold increased level of the encoded mRNA or protein, increased enzymatic activity of the CAR target gene, increased relative liver mass, increased release of a liver enzyme such as alanine aminotransferase into the serum, or increased DNA synthesis in the liver, as measured using the assays described herein. In another preferred embodiment, the increased induction is 2, 5, 10, or 20-fold greater in a mouse having wild-type CAR receptor activity than in a mouse having a mutation that reduces CAR receptor activity.
In one preferred embodiment, the candidate activator of a CAR receptor and a CAR receptor inverse agonist are administered to a mouse having a mutation that reduces CAR receptor activity or a mouse expressing a human CAR receptor. The level of induction of a CAR target gene is measured in the presence and absence of the candidate activator to determine whether the candidate activator effects an increase in the level of induction of the CAR target gene. The administration of the CAR receptor inverse agonist may decrease the initial level of induction of the CAR target gene and thus facilitate the detection of an increase in the induction mediated by the candidate activator.
The phrase "inhibit a CAR receptor" means a decrease in the rate of induction of a CAR target gene or transgene operably-linked to a promoter of a CAR target gene.
Preferably, the decreased induction of the CAR target gene or transgene in a mouse results in a 2, 5, 10, or 20-fold decreased level of the encoded mRNA, protein, enzymatic activity, relative liver mass, release of a liver enzyme into the serum, or DNA synthesis in the liver, as determined using the assays described herein. In another preferred embodiment, the decrease in the level of induction is 2, 5, 10, or 20-fold greater in a mouse having wild-type CAR receptor activity than in a mouse having a mutation that reduces CAR receptor activity.
In one preferred embodiment, the candidate inhibitor of a CAR receptor and a CAR receptor agonist are administered to a mouse having a mutation that reduces CAR
receptor activity or a mouse expressing a human CAR receptor. The level of induction of a CAR target gene is measured in the presence and absence of the candidate inhibitor to determine whether the candidate inhibitor effects a decrease in the level of induction of the CAR target gene. The administration of the CAR receptor agonist may increase the initial level of induction of the CAR target gene and thus facilitate the detection of a decrease in the induction mediated by the candidate inhibitor.
The phrase "having wild-type CAR receptor activity" or "having naturally-occurring CAR receptor activity" means having a substantially identical activity to that of a naturally-occurnng murine or human CAR receptor. By "substantially identical," as used herein, is meant at least 80, 90, 95, 99, or 100% of the activity of a naturally-occurring CAR receptor. The ability of a CAR receptor to induce a CAR target gene or a transgene operably-linked to a CAR responsive promoter may be routinely measured using assays for the encoded mRNA, protein, or enzymatic activity or assays for relative liver mass, a liver enzyme released into the serum, or DNA synthesis.
As used herein, "modulate the metabolism" refers to an increase or decrease in the rate of a CYP-catalyzed reaction of a compound, such as the oxidation of the compound.
For example, the rate of metabolism of the compound may be measured as the rate of formation of the oxidized product or the formation of a subsequent product generated from the oxidized intermediate. Alternatively, the rate of metabolism may be represented as the half life or rate of disappearance of the initial compound or as the change in toxicity or activity of the initial compound or a metabolite generated in a CYP-dependent , manner from the initial compound. For example, a second compound is said to modulate the metabolism of a first compound if the half-life, toxicity, or activity of the first compound is increased or decreased in the presence of the second compound.
Preferably, the change in the half-life, toxicity, or activity of the first compound or a metabolite of . the first compound is at least 25, 50, 100, 200, 500, or 1000% of the corresponding half-life, toxicity, or activity in the absence of the second compound. In another preferred embodiment, the change in the half-life, toxicity, or activity is at least 2, 5, 10, or 20-fold greater in a mouse having wild-type CAR receptor activity than in a mouse having a mutation that reduces CAR receptor activity. In various preferred embodiments, a second compound mediates a change of at least 2, 5, 10, or 20-fold in the magnitude of the half-life, activity, or toxicity of a first compound or a metabolite of the first compound, as measured in any of the assays described herein.
The half-life may be measured by determining the amount of the compound present in samples taken from the mouse at various time points; the amount of the compound may be quantified using standard methods such as high-performance liquid chromatography, mass spectrometry, western blot analysis using compound specific antibodies, or any other appropriate method. In preferred embodiments, a reaction required for the toxicity or activity of the first compound or a metabolite of the first compound (such as the reaction of an activated metabolite with DNA, RNA, or protein) is at least 25, 50, 100, 200, 500, or 1000% of the corresponding rate in the absence of the second compound. The toxicity of the first compound or a metabolite of the first compound may also be measured by determining the relative liver mass, amount of a liver enzyme released into the serum, or rate of DNA synthesis in the liver of a mouse. It is also contemplated that the rate of a reaction catalyzed by another enzyme involved in xenobiotic metabolism that is downstream of a CAR receptor may also be increased or decreased. In one preferred embodiment, the second compound modulates the metabolism of the first compound by activating or inhibiting a CAR receptor.
As used herein "activity of a compound" refers to a biological effect mediated by a compound. Examples of possible activities of compounds include binding to other molecules, modulation of a binding interaction between molecules, modulation.of the rate of catalysis of an enzyme, induction of physiological or behavioral changes, or any other therapeutically relevant activity of a compound.
As used herein "physiological effect" means a toxic effect, an activity, or the modulation of the expression of a CAR target gene mediated by a compound, as~
described above. For compounds that are metabolized to form a metabolite that has a different level of toxicity or activity as the initial compound, the physiological effect of the compound may also be measured by determining the half-life of the compound.
The term "promoter" refers to a minimal sequence sufficient to direct transcription of an operably-linked gene. The promoter may also be operably-linked to 5' regulatory sequences that modulate the transcription of the gene.
Brief Description of the Drawings Figure lA is a schematic illustration of the targeted disruption of the mouse CAR
gene. Boxes represent exons. Exons 2 and 3 (hatched boxes) contain the DNA
binding domain. Homologous recombination resulted in replacement of Exons 1 and 2 with the /3-gal and iZeo resistance genes. Restriction enzyme sites for Apa I (A), Hind III (H3), Not I (N), Sal I (S), and Xba I (X) are indicated.
Figure 1B is a picture of a gel showing genotype analysis by Southern blotting.
Genomic DNA from tail samples was digested with Hind III and hybridized with the 3' probes. The 10 Kb and 15 Kb bands were generated from wild-type and mutant alleles, respectively.
Figure 1C is a picture of a gel showing Northern blot analysis. The murine CAR
cDNA was used as a probe to determine the level of CAR mRNA expressed in liver of wild-type and CAR +/- or -/- animals.
Figure 2A is a picture of a representative Northern blot of xenobiotic activation of the CYP2B 10 gene by CAR in a liver sample. Mice (8-10 weeks old, 3 mice per treatment) were treated with corn oil (CO) for 6 or 24 hours, PB for 24 hours, or TCPOBOP for 6 hours.
Figure 2B is a series of photographs of in situ hybridization of a piece of small intestine from mice treated with PB or TCPOBOP for 3 days. The in situ hybridization was performed with a [35S]-labeled antisense CYP2B 10 riboprobe. The number of grains per cell is not significantly different in the CAR -/- animals, with or without xenobiotic treatments. The number of grains per cell is approximately two-fold higher in the untreated wild-type mice than in the CAR receptor knockout mice, and the number of grains per cell in the xenobiotic-treated wild-type mice is approximately two-fold higher than in the untreated wild-type mice.
Figure 3A is a bar graph showing the liver enlargement and hepatocyte proliferation by PB or TCPOBOP. Mice (8-10 weeks old) were treated with PB or TCPOBOP for 3 days, and then both liver mass and body weight were measured.
The data is presented as percentage of liver mass relative to total body weight.
Figure 3B is a set of pictures of PB-treated, TCPOBOP-treated, or control mice that were treated with BrdU for two hours before their liver tissues were harvested. The representative microphotography illustrates the presence of BrdU-positive hepatocytes only in PB- or TCPOBOP-treated wild-type animals.
Figure 4 is a bar graph showing the effect of PB or TCPOBOP on cocaine-mediated hepatotoxicity, measured as serum alanine aminotransferase (ALT) activity. Male mice were pretreated with PB or TCPOBOP for three days. Twenty-four hours after the last dose, one injection of cocaine was given to the animals.
Blood was drawn 20 hours after cocaine treatment for determination of serum ALT
activity.
Figures SA and 5B are pictures of representative Northern blots of xenobiotic activation of the murine CYP3A11 gene by murine CAR in a liver sample. Mice (8-weeks old, 3 mice per treatment) were treated with corn oil (CO) for 6 or 24 hours, PB
for 24 hours, or TCPOBOP for 6 hours.
Figure 6 is a schematic illustration of the transgene construct used to generate mice expressing human CAR. This transgenic construct contains the liver specific, albumin promoter operably linked to the cDNA sequence for human CAR. To enhance the expression and stability of human CAR transcripts, a region from an abundantly expressed gene, rabbit (3-globin, and the polyadenylation (poly A) sequence from bovine growth hormone were also added to this construct.
Figures 7A and 7C are pictures of representative Southern blots of genomic DNA
from mice generated using the human CAR transgene construct. The lanes containing DNA that bound to the human CAR probe are labeled with as asterisk.
Figure 7B is a representative gel showing the PCR amplification of genomic DNA
from mice generated using the human CAR transgene construct. The production of a PCR product using primers specific for human CAR confirmed the results of the Southern blot analysis in Figs. 7A and 7C. Based on these analyses, nine of the mice were identified as transgenic mice containing DNA encoding human CAR.
Figure 8 is a Northern blot illustrating expression of human CAR mRNA
transcripts in one of the humanized CAR mice lines (line 6210). Human CAR mRNA
was specifically expressed in the liver.
Figure 9 is a bar graph illustrating the induction of a CAR reporter gene in HepG2 cells transiently transfected with the human CAR transgenic construct illustrated in Fig.
6. These results indicate that this transgenic construct encodes functional human CAR
which can activate the expression of a reporter gene operably liked to a CAR
responsive promoter. These transfected cells may be used to screen candidate compounds to determine whether they activate or inhibit human CAR.
Figure 10 is a picture of a Northern blot illustrating induction of the CAR
target gene CYP2B 10 by PB, but not by TCPOBOP, in humanized CAR mice lacking a functional murine CAR gene. Animals with the indicated genotypes (with, "+,"
or without, "-," the line 6210 hCAR transgene, and heterozygous, "-/+," or homozygous, "-/-," for the murine CAR gene mutation) were treated with PB or TCPOBOP fox 24 hours.
Total RNA was prepared from the livers of the individual mice, and CYP2B 10 mRNA
expression was assessed by Northern blotting.
Figures 11A-11D demonstrate that CAR activation induces APAP toxicity. CAR
(+/+) or CAR (-/-) animals pretreated with phenobarbital (PB), TCPOBOP (TC), or vehicle alone were administered a 250 mg/kg dose of APAP by intraperitoneal injection (n=5-7 per treatment group) (Fig. 11A). Serum was collected and alanine aminotransferase (ALT) levels were measured after 24 hours. Liver sections from different treatments were examined by histological staining (Fig. 11B). Liver samples from all treated animals were analyzed but only representative histology is presented. PB
or TCPBOBP pretreated livers from wild-type animals showed extensive hepatic centrilobular necrosis as indicated by arrows. CAR (+/+) or CAR (-/-) animals were pretreated with PB, TCPOBOP (TC), or vehicle alone for three days (Fig. 11C).
Total liver RNA was prepared and subjected to Northern blot analysis with indicated probes.
Liver samples treated as indicated were collected two hours after APAP and GSH
levels were measured (n=5-7) (Fig. 11D). The hepatic GSH levels in the PB and TC
pretreated wild-type animals were significantly different from those of both the CAR
(+/+) animals injected with vehicle alone (p=0.011 and p=0.0046, respectively) and CAR (-/-) animals pretreated with PB and TC (p=0.034 and p=0.026, respectively).
Figures 12A and 12B show that CAR (-/-) mice are resistant to APAP toxicity.
CAR (+/+) and CAR (-/-) animals were given 250mg/kg, 500 mg/kg, or 800mg/kg doses of APAP, respectively (Fig. 12A). Blood samples were collected 5 or 24 hours later and serum alanine arninotransferase (ALT) levels were measured (n= 5-7). At the dose of 500 mg/kg and 800mg/kg, CAR (-/-) animals were significantly less sensitive than wild-type animals to APAP toxicity (with 500mg/kg, p=0.019 for five hours and p=
0.026 for 24 hours; with 800 mg/kg, p=0.016 for five hours and p=0.0008 for 24 hours).
Total liver RNA was prepared from 500 mg/kg APAP treated CAR (+/+) and CAR (-/-) livers at 0 and 2 hours as indicated. Total RNA (10 fig) from different samples was subjected to Northern blot analysis with indicated probes (Fig. 12B). The same blot was stripped and re-probed with (3-actin as loading control. Quantitation of mRNA levels by densitometry showed that the induction of CYP1A2, CYP3A11, and GSTPi was approximately 2.8, 4.4 and 3.9 fold, respectively.
Figure 13 illustrates the sensitivity of CAR humanized mice to APAP. CAR
humanized mice (n=3) were pretreated with phenobarbital (PB) or vehicle alone for three days and administered a 250 mg/lcg dose of APAP by intraperitoneal injection.
Serum was collected and alanine aminotransferase (ALT) levels were measured after 24 hours.
Figures 14A and 14B illustrate the hepatoprotection by androstanol treatment.
CAR (+/+) or CAR (-/-) animals were given a 500 mg/kg dose of APAP by intraperitoneal injection, either with or without an additional injection of androstanol (An, 100mg/kg) 1, 3, or 5 hours later (Fig. 14A). Serum alanine aminotransferase (ALT) levels were measured 24 hours later (n=5-7). At one and three hours, CAR (+/+) animals showed significantly lower ALT levels than CAR (-/-) animals (both p<0.0001).
Liver sections from the same animals 24 hours after different treatments as indicated were stained with hematoxylin and eosin (Fig. 14B). Arrows indicate areas of hepatic necrosis.
Figure 15A is a picture of the histological staining of liver sections after two hours of 500 mg/kg APAP treatment. Liver sections from animals injected with 500 mg/kg APAP for 0 or 2 hours were stained with hematoxylin and eosin. Arrows indicate initial stages of necrosis surrounding central veins. The same liver samples were collected and GSH levels were measured at 0 and 2 hours as indicated. Figure 15B is a graph of these GSH levels. CAR (-/-) animals were resistant to GSH depletion compared to CAR
(+/+) animals (p=0.032).
Figure 16 is a picture of a Northern blot indicating induction of genes by APAP.
CAR humanized mice (n=3) were treated with 500 mg/kg APAP or vehicle. After two hours, total liver RNA was prepared and subjected to Northern blot analysis with indicated probes. Quantitation of mRNA levels by densitometry indicated that the induction of CYP1A2, CYP3A11, and GSTPi was 1.8, 2.4 and 1.8 fold, respectively.
Figure 17 is a bar graph comparing the hepatoprotective effects of androstanol and N-acetylcysteine. Wild-type mice (n=3 per group) were administered a 500 mg/kg dose of APAP by intraperitoneal injection. One to three hours later, as indicated, androstanol (100 mg/leg) or N-acetylcysteine (1 g/kg) were given by intraperitoneal injection. Serum samples from each group were pooled, and ALT levels were measured 24 hours after APAP administration.
Figure 18 is the nucleic acid sequence (SEQ ID NO: 25) and deduced amino acid sequence (SEQ ID NO: 2) of a human CAR receptor (WO 99/15,555). Preferably, the DNA binding domain of human CAR includes amino acids 11-75 or a DNA binding fragment thereof. Preferably, the gene activation and/or ligand binding domain of human CAR includes amino acids 76-348 or a gene activating and/or ligand binding fragment thereof (WO 93/17,041). In preferred embodiments, a humanized CAR mouse expresses a human CAR protein that includes or that consists of a DNA binding domain, gene activation domain, and/or ligand binding domain, or fragment.thereof.
Figure 19 is the nucleic acid (SEQ ID NO: 26) and deduced amino acid sequence (SEQ >D NO: 1) of a murine CAR receptor, mCARl (WO 99/15,555). Preferably, the DNA binding domain of murine CAR includes amino acids 21-86 or a DNA binding fragment thereof. Preferably, the gene activation and/or ligand binding domain of human CAR includes amino acids 182-358 or a gene activating and/or ligand binding fragment thereof.
Figure 20A is a schematic representation of the sequences of mCARl, mCAR2, and human CAR. The sequence of mCARl (Genbank accession number AF009327) is shown, and differences between mCARl and human CAR and also the mCAR2 variant (Genbank accession number AF900328) are indicated. The DNA binding domain is in bold. Dots indicate residues not present in human CAR. The positions of the introns in the mCAR gene are indicated. The first intron is 5 nucleotides upstream of the ATG
encoding the first methionine. Introns that fall within a codon are indicated after the corresponding amino acid (WO 99115,555).
Figure 20B is a schematic representation indicating comparisons of mCARl to related members of the nuclear hormone receptor superfamily. The DNA binding, hinge, and ligand binding/dimerization domains of mCAR1 are indicated, and the percent identity of the analogous domains of related proteins is indicated. VDR is the human vitamin D receptor, xONR is a Xenopus laevis orphan receptor (Smith et al., Nucl. Acids Res. 22:66-71, 1994), and EcR is the Drosophila melanogaster ecdysone receptor (WO
99/15,555).
Figure 21 is a diagram indicating the positions of the mCAR introns, 5' untranslated and 3' untranslated regions of the primary transcript, and the positions of various portions of the transcript and protein. For the 3' extended transcripts, the 3' untranslated region extends into the 3' flanking region. The ~th exon deleted in mCAR2 is indicated by a star (WO 99/15,555).
Detailed Description of the Invention The present invention is directed to compositions and methods relating to the function of the nuclear hormone receptor, CAR, particularly in human biological processes involving xenobiotic metabolism. Accordingly, the present invention provides transgenic animals comprising a mutation in a CAR receptor. Particularly, the present invention provides transgenic animals deficient of or lacking in the CAR gene, and comprising a reduction or absence of CAR receptor activity. The present invention also provides transgenic animals expressing a human CAR receptor. The transgenic animals of the present invention may be used, for example, in methods for identifying CAR
ligands, including compounds that induce the activation of CAR target genes, and measuring the toxicity and half-life of such compounds. The present invention also provides methods which allow for the identification of compounds that activate CAR receptors and are potentially toxic to mammals (e.g., humans), as well as compounds that inhibit CAR
receptors and reduce the toxicity or CYP-mediated metabolism of a pharmaceutically active compound (e.g., acetaminophen) administered to a mammal.
CAR Receptor Knockout Animals The present invention provides transgenic animals which are deficient'of or lacking in a CAR gene ("CAR knockout"): . The transgenic animals of the present invention include transgenic animals that are heterozygous (+/-) and homozygous (-/-) for a mutation in the CAR gene. In one aspect, the transgenic animals of the present invention comprise a reduction or absence of native or naturally-occurring CAR
receptor activity. In a preferred aspect, the transgenic animals of the present invention are mice.

Various methods known in the art may be used to produce CAR knockout mice of the present invention. For example, an appropriate targeting vector may be produced using standard methods known in the art (see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory MafZUal, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; E.N. Glover (eds.), 1985, DNA Clo~eing: A
Practical Approach, Volumes I and II; M.J. Gait (ed.), 1984, Oligonucleotide Syfithesis;
B.D.
Hames & S.J. Higgins (eds.), 1985, Nucleic Acid Hybridization; B.D. Hames &
S.J.
Higgins (eds.), 1984, Transcription and Translation; R.I. Freshney (ed.), 1986, Animal Cell Culture; Immobilized Cells and Enzymes, IRL Press, 1986; B. Perbal, 1984, A
Practical Guide To Molecular Cloning; F.M. Ausubel et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). For example, the targeting vector may be prepared in accordance with conventional ways, where sequences may be synthesized, isolated from natural sources, manipulated, cloned, ligated, subjected to ira vitro mutagenesis, primer repair, or the like. At various stages, the joined sequences may be cloned, and analyzed by restriction analysis, sequencing, or the like.
Once an appropriate targeting vector has been prepared, the targeting vector may be introduced into an appropriate host cell using any method known in the art.
Various techniques may be employed in the present invention, including, for example:
pronuclear microinjection; retrovirus mediated gene transfer into germ lines; gene targeting in embryonic stem cells; electroporation of embryos; sperm-mediated gene transfer; and calcium phosphate/DNA co-precipitates, microinjection of DNA into the nucleus, bacterial protoplast fusion with intact cells, transfection, polycations, e.g., polybrene, polyornithine, or the like (see, e.g., U.S. Patent No. 4,873,191; Van der Putten, et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152; Thompson, et al., 1989, Cell 56:313-321; Lo, 1983, Mol Cell. Biol. 3:1803-1814; Lavitrano, et al.,1989, Cell, 57:717-723).
Various techniques for transforming mammalian cells are known in the art (see, e.g., Gordon, 1989, Intl. Rev. Cytol., 115:171-229; Keown et al., 1989, Methods in Enzymology; Keown et al., 1990, Methods and Enzy»aology, Vol. 185, pp. 527-537;
Mansour et al., 1988, Nature, 336:348-352).
Successful insertion of the targeting vector by homologous recombination is typically detected by identifying cells for expression of the marker gene. In a preferred embodiment, the cells transformed with the targeting vector are subjected to treatment with an appropriate agent that selects against cells not expressing the selectable marker.
Only those cells expressing the selectable marker gene survive and/or grow under certain conditions. For example, cells that express the introduced neomycin resistance gene are resistant to the compound 6418, while cells that do not express the neo gene marker are killed by 6418.
Selected cells are then injected into a blastocyst (or other stage of development suitable for the purposes of creating a viable animal, such as, for example, a morula) of an animal (e.g., a mouse) to form chimeras (see, e.g., Bradley, A. in Teratocarcinomas and Ewbryoni.c Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, pp.
113-152 (1987)). Alternatively, selected ES cells can be allowed to aggregate with dissociated mouse embryo cells to form the aggregation chimera. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Chimeric progeny can be used to breed animals to generate heterozygous mice. Heterozygous mice can then be mated to produce homozygous mice.
Characterization and Analysis of CAR Receptor Knockout Miee In a preferred embodiment, CAR knockout mice of the present invention exhibit a decrease or absence of induction, expression or activity of a CAR target gene in response to a xenobiotic inducer. In a particular embodiment, CAR knockout mice of the present invention exhibit a decrease or absence of CYP enzyme induction, activity, or expression in response to a xenobiotic inducer, including PB and TCPOBOP.
More specifically, CAR knockout mice exhibited an absence in the expression of CYP2B 10 in response to either PB or TCPOBOP. The robust induction of expression of CYP2B 10 mRNA in response to either ~PB or TCPOBOP was detected in wild-type male or female mice but was absent in knockout animals (Fig. 2A). Similar results were obtained with two independent CAR knockout mouse lines. This requirement for CAR
was also demonstrated in the small intestine, using in situ hybridization. As indicated in Fig. 2B, either PB or TCPOBOP also induced CYP2B 10 expression in the small intestine in wild-type mice, but was not detected in CAR knockout mice. Similar to the induction of CYP2B 10, substantial induction of murine CYP3A 11 mRNA in response to PB
or TCPOBOP was observed in wild-type mice (Fig. 5A). In contrast, a negligible level of induction of CYP311 was detected in CAR -/- mice (Fig. 5B).
In another embodiment, CAR knockout mice of the present invention exhibit an absence of increased liver mass or no enlargement of the liver upon treatment with an xenobiotic inducer, including PB or TCPOBOP. More specifically, hyperplasia or hypertrophy in the liver in response to treatment with either phenobarbital or the more potent inducer TCPOBOP were not detected in the CAR knockout mice.
Acute treatments with PB-like inducers, particularly TCPOBOP, cause an up to 2-fold increase in liver mass relative to total body mass (See, e.g., Heubel et al., Bioehenz.
Pharmacol. 38:1293-1300, 1989). This hepatomegaly is thought to be a reflection of both cellular hypertrophy and mitogenesis. After three days of treatment with either PB
or TCPOBOP, an increase in liver mass was observed in wild-type mice. Whereas, CAR -/- mice subjected to the same PB or TCPOBOP treatments did not show any evidence of increased liver mass as observed in the wild-type mice (Fig. 3A).
In a further embodiment, CAR knockout mice of the present invention exhibit an absence of xenobiotic induction of DNA synthesis when treated with 5-bromodeoxyuridine (BrdU). As demonstrated in Figure 3B, xenobiotic induction of DNA synthesis revealed by increased incorporation of BrdU in the wild-type animals was completely absent in the CAR -/- animals.
In yet a further embodiment, CAR knockout mice of the present invention exhibit decreased metabolism of the CYP substrate zoxazolamine. Many studies have demonstrated that increased CYP enzyme activity results in increased metabolic inactivation of this muscle relaxant, which is reflected in decreased duration of zoxazolamine-induced paralysis. As demonstrated in Tables 1 and 2, pretreatment of wild-type animals with either PB or TCPOBOP significantly decreased the duration of paralysis. The duration of paralysis was substantially longer in untreated CAR
-/- mice than in wild-type mice, the paralysis was not affected by pretreatment with either PB or TCPOBOP. For example, wild-type control female mice were paralyzed more than hours, while wild-type xenobiotic-pretreated female mice were not paralyzed.
Among the CAR -/- females, two animals from each group of control, PB-treated, or TCPOBOP-treated animals died; the survivors were paralyzed for more than 12 hours.

Table 1. Increased duration of zoxazolamine-induced paralysis in male CAR (-/-) mice due to decreased metabolism of zoxazolamine CAR +/+ CAR -/-Control 2 hours . > 5 hours PB-treated < 20 minutes > 5 hours TCPOBOP-treated < 20 minutes > 5 hours Table 2. Increased duration of zoxazolamine-induced paralysis in female CAR (-/-) mice due to decreased metabolism of zoxazolamine CAR +/+ CAR -/-Control >12 hours died PB-treated not paralyzed died TCPOBOP-treated not paralyzed died In these experiments, mice were pretreated for three days with PB or TCPOBOP, after which time they were given a single intraperitoneal injection of zoxazolamine (300 mgikg). Paralysis time was recorded as the time when the mice were able to right themselves repeatedly.
Tn another embodiment, CAR knockout mice of the present invention are absent of liver damage when treated with cocaine in the presence of a xenobiotic inducer, such as PB or TCPOBOP. Preferably, CAR knockout mice of the present invention pretreated with PB or TCPOBOP do not exhibit acute hepatotoxicity or cocaine-mediated hepatotoxicity when injected with cocaine For example, treatment with PB-like inducers, such as PB or TCPOBOP also sensitizes animals to hepatotoxic effects of a number of compounds, including cocaine.. As shown in Fig. 4, treatment with either PB
or TCPOBOP resulted in a significant increase in serum levels of the liver enzyme alanine aminotransferase (ALT) as an acute response to cocaine administration.

Evidence of liver damage was not observed in CAR -/- animals.
In a further embodiment, CAR knockout mice exhibit a resistance to acetaminophen toxicity. As described below, inhibition of CAR activity by administration of the inverse agonist ligand androstanol one hour after acetaminophen treatment blocked hepatotoxicity in wild-type mice, but not in CAR null mice.
Functional Role of CAR Receptor The present invention demonstrates that CAR is essential for response to PB-like inducers of xenobiotic metabolism, and thus CAR functions as a xenobiotic receptor in vivo to mediate the response to PB-like inducers. CAR can therefore be added to the previously described peroxisome proliferator activated receptor a and the aryl hydrocarbon receptor as a primary determinant of the response of phase I
metabolic enzymes to foreign compounds. CAR is joined in this by its closest relative within the receptor superfamily, PXR/SXR, which has recently been shown to mediate response to a distinct group of xenobiotics (See, e.g.,~Jones et al., Mol. Endocrinol. 14:27-39, 2000;
Kliwer et al., Cell 92:73-82, 1998; Blumberg et al., GefZes Dev. 12:3195-3205).
Although both DNA binding specificity and xenobiotic responses of CAR and PXR/SXR
have been reported to overlap to some extent, no evidence for any compensatory effect of the latter was observed in the CAR knockout animals. Thus, it is now apparent that specific xenobiotics can induce specific metabolic responses by activating distinct receptors.
This mechanism may account for a large number of clinically significant drug-drug interactions in which the presence of one compound, such as phenobarbital, results in increased metabolism of another drug or foreign compound.
Differences in the levels of activation of xenobiotic receptors among individuals based on differences in exposure to specific xenobiotics may also explain the significant inter-individual variability of the levels of particular cytochromes. Consistent with the very low basal levels of mouse CYP2B 10, most humans have low or undetectable levels of CYP2B6, a human target of CAR activation. However, this enzyme is present at up to 100-fold higher levels in a subset of individuals. The results described here suggest that this variability could be the basis for the relatively rare, but clinically significant hepatotoxicity observed in a subset of individuals exposed to high levels of cocaine.
More generally, variations in CAR activity in response to the wide range of PB-like inducers may have significant impact on the metabolism of a wide range of pharmacologic agents and other foreign compounds. The CAR mice described herein facilitate the identification of compounds able to activate CAR in vivo, as well as enable the identification of additional, specific downstream target genes that mediate its effects.
CAR Target Genes Various CAR target genes are known in the art and may be applicable to the compositions and methods of the present invention. The CAR target genes include, but are not limited to cytochrome P450 genes, particularly, cytochrome P450 genes and other enzymes involved in xenobiotic metabolism (see, e.g., Waxman, Archives of .
Biocherrcistry and Biophysics 369, l, 1999; Kliewer et al., Science 284, 757-760, 1999).
Preferred CAR target genes include murine CYP2B 10 (SEQ 117 NO: 11, Accession No.
NM_009998) and human CYP2B6 (SEQ )D NO: 12, GenBank Accession No.
AC023172). Additional preferred CAR target genes include murine CYP3A11 (SEQ
>D
NO: 13, Accession No. NM_07818) and human CYP3A4 (SEQ ID NO: 14, Accession No. A34101).
CAR target genes may also include a CAR responsive promoter operably-linked to a reporter gene, such as human growth hormone, secreted alkaline phosphatase, chloramphenicol acetyl transferase, luciferase, green fluorescent protein, CYP2B6, or any other reporter gene (see, for example, Ausubel et al., Current Protocols in Molecular Biology, Chapter 9, John Wiley & Sons, New York, 2000). Examples of appropriate promoters include native CYP promoters, such as the CYP2B10 promoter (Gen Bank Accession No. U48732; Honkakoski et al., J. Biol. Chem. 271, 9746-9753, 1996) containing the previously described Phenobarbital response element (Honkakoski et al., Mol. Cell. Biol. 18:5652-5658, 1998), the CYP2B6 promoter (GenBank Accession No.
AC023172), the CYP3A11 promoter (Toide et al., Arch. Biochem. Biophys.
338(1):43-49, 1997), the CYP3A4 promoter (Accession No. AF185589), or synthetic promoter constructs in which DNA binding sites for CAR/RXR heterodimers are operably-linked to functional basal promoters (Tzameli et al., Mol. Cell. Biol. 20: 2951-2958, 2000).
The determination or measurement of the induction of CAR target genes by CAR
may be accomplished using standard methods known in the art. More particularly, the level of induction of the CAR target gene or transgene may be determined using standard assays for measuring the level of encoded mRNA or protein (see for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000).
Alternatively, an enzymatic activity of a CAR target gene, such as the 7-pentoxyresorufin O-dealkylase activity of the CYP2B 10 CAR target gene, may be measured (Pellinen et al..Hepatology 23:515-23, 1996). Examples of CAR target genes include CYP2B
10, a CYP2B6, CYP3A11, and CYP3A4; examples of CAR responsive promoters include the CYP2B10, CYP2B6, CYP3All, and CYP3A4 promoters and promoters operably-linked to DNA binding sites for CAR/RXR heterodimers. Alternatively, an increase in CAR
receptor activity can be assayed by determining an increase in liver mass relative to total body mass, an increase in release of a liver enzyme such as alanine aminotransferase into the serum, or an increase in DNA synthesis in the liver, using the assays described herein.
CAR-mediated induction may be measured in response to a number of xenobiotic compounds, including TCPOBOP.
Trans~enic Mice Expressing Human CAR Receptor A humanized CAR mouse expressing the human CAR receptor may be generated by any of several standard methods (see, for example, Ausubel et al. (Chapter 9), supra).
Preferably, the humanized CAR mouse lacks murine CAR. For example, a conventional transgenic animal expressing the human CAR gene from a promoter active in appropriate tissues, such as the liver or the intestine, may be generated. Also contemplated by the present invention is a humanized CAR mouse which expresses a region or portion of the humanized CAR receptor that is sufficient for binding of a ligand, including the human CAR ligand binding domain or a portion thereof (see, e.g., PCT Publication No.
WO
0171361).
Various promoters known in the art may be used for directing expression of the human CAR receptor. Examples of such promoters for expression of transgenes include, but are not limited to the zinc-inducible metallothionein promoter, the DEX-inducible tyrosine aminotransferase (TAT) promoter, the rifampicin inducible CYP3A4 promoter, the albumin promoter (Xie et al., Nature 406:435-439, 2000), the fatty acid binding protein (FABP), transthyretin (TTR) (Ye et al., Mol Cell Biol. 19:8570-8580, 1999), or CAR promoter. The FABP promoter is expressed in the liver and small intestine.
The TTR promoter is also well described and widely used promoter to achieve liver-specific expression of transgenes. Other regulatory sequences may be used to enhance expression of the human CAR receptor in a humanized CAR mouse.
The human CAR transgene may then be introduced into a homozygous CAR -/-mouse by conventional breeding (Pierson et al., Mol. Endocri~2ol. 14:1075-1085, 2000;
Slee et al., Proc Natl Acad Sci U S A. 96:8040-8045, 1999). In another possible method, the human CAR transgene may be injected into fertilized oocytes from homozygous CAR
-/- mice, directly generating the desired transgenic mice. In a third method, embryonic stem cells may be generated from a homozygous CAR -/- animal (Ausubel et al.
(Chapter 9), supra). Conventional homologous recombination techniques may then be used to replace the inactivated rnurine CAR gene with a functional human CAR receptor gene (Fiering et al., Methods Enzymol. 306:42-66, 1999). Since the CAR -/- animals contain the neo gene which confers resistance to 6418, another appropriate gene such as hygromycin may be used in the human CAR replacement construct to allow the selection of cells in which the human CAR gene has replaced the inactivated murine CAR
gene. In still another possible method, a functional human CAR receptor gene may be introduced into a homozygous CAR -/- mouse using gene therapy and a promoter active in appropriate tissues, such as the promoters described above, and contained in an adenoviral, adeno-associated viral, retroviral, lentiviral, herpes viral, nonviral,. or any other suitable vector (see, for example, Sarkar et al., Hum Gene Ther. 11:881-894, 2000;
Goddard et al., Gefze Ther. 4:1231-1236, 1997).
If desired, mice expressing human CAR, but lacking mouse CAR can be generated by mating humanized CAR mice with the CAR -/- mice described above.
Alternatively, ES cells or embryos from humanized CAR mice can be genetically modified as described above to disrupt the endogenous mouse CAR gene.
It is known that the ligand binding domains of human and mouse CAR genes differ somewhat in amino acid sequence, and that these two proteins respond differently to some activators. In particular, TCPOBOP is an agonist ligand for the murine CAR
receptor, but not the human CAR receptor (Tzameli et al., supra). Similarly, clotrimazole is an inverse agonist for the human, but not the murine, CAR
receptor (Moore et al., supra). As a result, CAR -/-'knockout mice expressing a functional human CAR receptor also provide useful models for drug screening since their response to drugs or other xenobiotic compounds should be based on the human rather than the murine CAR receptor. Such "humanized" CAR mice allow for the identification of CAR
ligands, including compounds, such as those in clinical development, with potentially undesirable effects in humans.
In one aspect, transgenic mice of the present invention expressing human CAR
express only human CAR in the liver. In, a particular embodiment, transgenic mice expressing human CAR exhibit an induction of a xenobiotic response, particularly, an induction of a CAR target gene, such as CYP2B 10 mRNA in response to PB. In another particular embodiment, the transgenic mice expressing human CAR do not demonstrate or exhibit an induction of an xenobiotic response, such as activation of a CAR
target gene in response to TCPOBOP.
Human CAR-Mediated Toxicity As described in the examples below, CAR was determined to function as a key regulator of acetaminophen metabolism and hepatotoxicity. Both known CAR
activators and high doses of acetaminophen induced expression of three acetaminophen metabolizing enzymes in wild-type, but not CAR null mice. Additionally, CAR
null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol one hour after acetaminophen treatment blocked hepatotoxicity in wild-type, but not CAR null mice. These results demonstrate that inhibitors of CAR receptor expression and/or activity are useful for the treatment of any adverse-side effects associated with acetaminophen or other hepatotoxic agents in mammals (e.g., humans).
In particular, overdoses of acetaminophen (APAP, also known as 4'-hydroxyacetanilide, N-acetyl-p-aminophenol and paracetamol) are the leading cause of hospital admission for acute liver failure in the United States. Ingestion of amounts of APAP only 2-3 fold above the maximum daily recommended dose can cause hepatotoxicity and higher doses result in centrilobular necrosis that is potentially fatal.
The basis for this toxicity has been well studied. Particularly at high doses, cytochrome P450 enzymes, especially CYP1A2, CYP2E1, and isoforms of CYP3A, convert APAP
to a reactive quinone form, N-acetyl p-benzoquinone imine (NAPQI) that covalently binds to cellular macromolecules and also causes production of reactive oxygen species. At subtoxic doses, NAPQI is inactivated by glutathione S transferases (GSTs) via conjugation with reduced glutathione (GSH), but NAPQI accumulates when GSH
levels are depleted. Among the numerous GST enzymes, the GSTPi isoforms are particularly effective at NAPQI inactivation. Their importance in APAP toxicity was confirmed by the unexpected demonstration that knockout mice lacking both GSTPi isoforms are relatively resistant to APAP hepatotoxicity due to a decreased rate of GSH
depletion (Henderson et al., Proc Natl Acad Sci U S A 97:12741-12745, 2000).
APAP toxicity is increased in both humans and rodents by pretreatment with various inducers of CYP gene expression including ethanol, an inducer of CYP2E
and 3A
isoforms (Kostrubsky et al., Toxicol Appl Pharnaacol 143:315-323, 1997 and Sinclair et al., Biochem Pharmacol 55:1557-1565, 1998) and PB, a well known inducer of CYP2B, 3A and other isoforms (Pirotte, Ann Intern Med 101:403, 1984 and Burk et al., Res Commun Chem Pathol Pharmacol 69:115-158, 1990).
Assays for CAR Receptor Activity .
The homozygous CAR -/- animals described herein are useful for drug metabolism assays since these animals alloy for the identification of CAR
ligands and drugs or other xenobiotic compounds that induce expression of CYP2B 10, CYP3A11, or other CAR target genes in wild-type, but not CAR -/- animals. The detection of CYP2B10 or CYP3A11 induction may be performed by any of several assays, including assays of CYP2B10 or CYP3Al1 protein levels (for example, by Western blot analysis), mRNA levels (for example, by Northern blot analysis), or enzymatic activity (for example, my measuring 7-pentoxyresorufin O-dealkylase enzymatic activity as described, for example, in Pellinen et al: (Hepatology 23:515-23, 1996)).
Alternatively, an increase in liver mass relative to total body mass or an increase in DNA
synthesis in the liver may be measured as described herein. Similar assays for other CAR
target genes may also be used.
Moreover, as alternatives to assays involving endogenous murine CAR target genes, assays may be conducted to measure appropriate reporter transgenes inserted by any standard technique (for example, those techniques described above) into wild-type mice, CAR -l- mice, humanized CAR mice, mice lacking the gene encoding the receptor related to CAR (known as SXR, PXR or by other names (Kliewer et. al., Cell 92:73 - 82, 1998; Blumberg et al., Genes Dev. 12:3195-3205, 1998)), or any other appropriate strain.
These reporter transgenes consist of a CAR responsive promoter operably-linked to an easily measured reporter gene. Examples of appropriate promoters include native CYP
promoters such as the CYP2B 10 promoter containing the previously described phenobarbital response element (Honkakoski et al., supra), the CYP2B6 promoter, the CYP3A11 promoter, the CYP3A4 promoter, or synthetic promoter constructs in which DNA binding sites for CAR/RXR heterodimers are linked to functional basal promoters (Tzameli, et al., supra). Examples of appropriate reporter genes include, without limitation, human growth hormone, secreted alkaline phosphatase, luciferase, green fluorescent protein, chloramphenicol acetyl transferase, CYP2B6, CYP3A11. and any other reporter gene (see, for example, Ausubel et al. (Chapter 9), supra). The assays for CAR target genes involve standard procedures (see, for example, Ausubel et al.
(Chapter 9), supra) and may be based on appropriate samples from the mice, such as liver or serum samples. Alternatively, hepatocytes or other appropriate cell types may be harvested from the animals and propagated. Compounds may be administered to these cells to determine whether the compounds effect a change in expression of CAR target genes or reporter transgenes.
Candidate compounds may also be tested for their ability to activate or inhibit human CAR in cell based assays using cells that have been transiently or stably transfected with a transgenic construct encoding human CAR. For example, the Alb-hCAR transgenic construct illustrated in Fig. 6 which encodes human CAR under the control of the albumin promoter was used to transiently transfect a human hepatoma derived HepG2 cell line. The HepG2 cell lines contains a plasmid with a previously described reporter construct, denoted betaRAREluciferase, which contains a luciferase reporter gene under the control of a CAR responsive promoter (Forman et al., Nature 395(6702):612-5, 1998). As illustrated in Fig. 9, transfection of the cells with the Alb-hCAR transgene construct resulted in a 5.2-fold greater level of luciferase reporter gene expression than transfection of the cells with the corresponding control construct which lacks the human CAR coding sequence. This result indicates that this transgenic construct encodes functional human CAR which can activate the expression of a reporter gene operably liked to a CAR responsive promoter. The cells transfected with the Alb-hCAR construct can be used to identify candidate compounds that increase the expression of the luciferase reporter gene as activators of human CAR and to identify candidate compounds that decrease the expression of the luciferase reporter gene as inhibitors of human CAR.
Therapeutic Methods and Routes of Administration The invention also features methods for preventing, stabilizing, or treating toxicity associated with CAR-mediated metabolism of a xenobiotic by administering an inhibitor of CAR receptor expression and/or activity to a mammal (e.g., a human).
Accordingly, in one such aspect, the invention features a method for preventing, stabilizing, or treating an adverse effect or symptom associated with one or more hepatotoxic agents (e.g., acetaminophen and/or phenobarbital) in a mammal (e.g., a human) by administering to a mammal (for example, a human) one or more compounds that inhibit the expression and/or activity of a CAR receptor in the mammal.
Preferably, the compound decreases the number of liver cells that are killed by the hepatotoxic agent by at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% compared to a vehicle control. In preferred embodiments, compound decreases the serum alanine aminotransferase activity of a mammal treated with the hepatotoxic agent by at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% compared to a vehicle control. In some embodiments, the level of reduced glutathione decreases by less than 20, 30, ~40, 50, 60, 70, 80, 90, or 95% in a mammal treated with both the compound and the hepatotoxic agent. Preferably, the compound decreases the expression or an activity of CAR (e.g., the induction of a CAR
target gene) by at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% compared to the corresponding expression or activity of CAR in the presence of the hepatotoxic agent, but in the absence of the compound. Exemplary hepatotoxic agents include compounds whose metabolism is induced by human CAR.
With respect to the therapeutic methods of the invention, it is not intended that the administration of compounds to a mammal be limited to a pauticular mode of administration, dosage, or frequency of dosing; the present invention contemplates all modes of administration, including oral, intraperitoneal, intramuscular, intravenous, intraarticular, intralesional, subcutaneous, or any other route sufficient to provide a dose adequate to prevent or treat an infection. One or more compounds may be administered to the mammal in a single dose or multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, one week, one month, one year, or ten years. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. If desired, conventional treatments (e.g., treatments that reduce liver toxicity such as N-acetylcysteine) can be used in combination with the compounds of the presentinvention.
Suitable earners include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The composition can be adapted for the mode of administration and can be in the form of, for example, a pill, tablet, capsule, spray, powder, or liquid. In some embodiments, the pharmaceutical composition contains one or more pharmaceutically acceptable additives suitable for the selected route and mode of administration. These compositions may be administered by, without limitation, any parenteral route including intravenous, intra-arterial, intramuscular, subcutaneous, intradermal, intraperitoneal, int>:athecal, as well as topically, oraaly, and by mucosal routes of delivery such as intranasal, inhalation, rectal, vaginal, buccal, and sublingual. In some embodiments, the pharmaceutical compositions of the invention are prepared for administration to the mammal in the form of liquids, including sterile, non-pyrogenic liquids for injection, emulsions, powders, aerosols, tablets, capsules, enteric coated tablets, or suppositories.
Exemplary mammals include humans, primates such as monkeys, animals of veterinary interest (e.g., cows, sheep, goats, buffalos, and horses), and domestic pets (e.g., dogs and cats).
The following examples are intended only to illustrate the present invention and should in no way be~construed as limiting the subject invention.
Examples Example 1: Targeting Vector Construction To construct the targeting vector for the CAR locus, an Xba I - Eag I fragment containing the nuclear localized ~i-galactosidase gene from vector pPD 46.21 was subcloned into the Xba I and EcoR I sites of the pGKneo plasmid. DNA from AB 1 ES
cells was used to amplify CAR genomic fragments for both 5' and 3' arms. For the 5' arm, a 3 kb CAR promoter fragment was cloned into the Apa I and Xba I sites.
.For the 3' arm, a 5 kb fragment spanning exons 3 to 9 was cloned into the Sal I and Not I
sites. The primers for the 5' arm were 5'-gcgcgcgggccctggcatacattaacacaaacacatacatat-3' (SEQ >D
NO.: 3) and 5'-gcgcgctctagaaggacccagactctggacccagggcaaaga-3' (SEQ ID NO: 4).
The primers for the 3' arm were 5'-gcgcgcgtcgacaggtgaagtgcttctccccaacagaaacaa-3' (SEQ ID
NO: 5) and 5'-gcgcgcgcggccgctgtcctgggagcagcctctgcagccgct-3' (SEQ IL7 NO: 6).
Example 2: Generation of CAR Receptor Knockout Mice AB 1 ES cells (107) were electroporated with 25 ~,g targeting construct in 0.9 ml PBS using a Bio-Rad Gene Pulser (500 ~,F, 230 V). The cells were then plated on one or two 10-cm plates containing a monolayer of irradiated STO feeder cells. About twenty-four hours later, the cells were subjected to 6418 selection (350 pg/ml, Gibco) for 9 days.
Resistant clones were analyzed by Southern blotting after Hind III digestion, using the 3' probe indicated in Fig. 1A (Fig. 1B). The primers for the 3' probe were 5'-ggacaacctcagcccacagtgatgc-3' (SEQ ID NO: 7) and 5'-tcctttggttaccacctgactctgc-3' (SEQ ID NO: 8). Two positive clones were expanded and injected into C57BL/6 blastocysts. Male chimeras were back crossed to C57BL/6 female mice.
Heterozygotes were determined by Southern blotting and intercrossed to generate homozygotes.
Example 3: Analysis of CAR and CAR Knockout Mice To assess the functional role of CAR, two independent mouse lines were generated in which a promoter proximal segment of the CAR gene, including a portion of the DNA
binding domain, was replaced by the coding region for (3-galactosidase (Fig.
1A). These [3-galactosidase "knockin" animals were unable to express CAR mRNA (Fig. 1C).
This loss of CAR expression did not result in any overt phenotype; homozygous CAR -/-animals were born at expected Mendelian frequency, and both male and female CAR -/-animals were fertile.
Example 4: Animal Treatment At least three mice between about 8-10 weeks old were used for each treatment.
Mice were pretreated by intraperitoneal injection with corn oil, PB (100 mg/lcg, Sigma), or TCBOPOP (3 mg/kg) for the indicated time. For a three day PB treatment, mice were injected intraperitoneally three times with PB, at one injection per day.
Example 5: Zoxazolamine Paralysis Test Mice pretreated with corn oil, PB, or TCPOBOP were given a single intraperitoneal injection of zoxazolamine (300 mg/kg, Sigma) approximately 24 hours after the last dose of PB. Mice were placed on their backs, and the paralysis time was defined as the time required for the animal to regain sufficient consciousness to right itself repeatedly (Liang et al., Proc. Natl. Acad. Sci. U S A, 93:1671-6, 1996).
Example 6: Cocaine Treatment and Alanine Aminotransferase (ALT) Assay Male mice pretreated with corn oil, PB, or TCPOBOP were injected intraperitoneally with cocaine HCl (30 mg/kg) 24 hours after the last dose of PB. The mice were anaesthetized 24 hours after cocaine treatment. Blood was drawn from the eye for determination of serum ALT activity.
Example 7: RNA Analysis Approximately 20 pg of total RNA from individual mouse livers was subjected to Northern blot analysis (Fig. 1C). A mouse CAR cDNA probe was used to reveal the absence of CAR transcripts in the CAR~nul1 mice. Probes for CYP2B10 were prepared by RT-PCR with mouse liver total RNA using Superscript One-step RT-PCR System (Life Technologies). PCR primers were 5'-ccgcctctagaagtcaacattggttagac-3' (SEQ
ID
NO: 9) and 5'-ccgccggatcccacactaagcctcataat-3' (SEQ ID NO: 10). For in situ hybridization, small intestine tissue was cross sectioned at 7 ~,M thickness.
Slides were subjected to in situ hybridization with a .[35S]-labeled CYP2B 10 antisense probe., To prepare the probe, the CYP2B 10 RT-PCR product was subcloned into the Xba I
and Barn HI sites of Bluescript7 SK(-) phagemid (Stratagene). The plasmid was linearized with Xba I. T7 RNA polymerase was used to synthesize [35S]-labeled antisense probes.
Example 8: Hepatocyte Proliferation .
To determine the proliferation of hepatocytes following PB or TCPOBOP
treatment, mice pretreated with corn oil, PB, or TCPOBOP received a single intraperitoneal dose of BrdU/FdU (2 m11100 g, Amersham). Mice were sacrificed approximately 2 hours after BrdU administration. BrdU incorporation was determined using a mouse anti-BrdU monoclonal antibody (DAKO Corporation) and Vectastain ABC Kit (Vector Laboratories Inc.) (Fig. 3B), using standard procedures.
Example 9: Human CAR Transgene Construct To generate transgenic mice expressing a human CAR receptor, the transgene construct illustrated in Fig. 6 was used. The transgenic construct contained the coding sequence for a human CAR receptor operably linked to the liver specific, albumin promoter. Additionally, a region from an abundantly expressed gene, rabbit (3-globin, was added between the promoter and the CAR receptor coding sequence to enhance the expression of the human CAR receptor. The polyadenylation (poly A) sequence from bovine growth hormone was also added downstream of the human CAR receptor coding sequence. The bovine growth hormone is a strong poly A sequence that ensures termination of transcription and stabilization of the mRNA transcripts.
Furthermore, the use of a heterologous poly A sequence eliminates the requirement to isolate the genomic sequence which corresponds to the endogenous poly A sequence of human CAR.
The transgenic human CAR construct was generated from several previously described plasmids. To generate the starting vector, a BamHI-EcoRI 640 base pair fragment consisting of part of exon 2, intron 2, and exon 3 of the rabbit ~3-globin gene [nucleotides 551-1190 from Entrez accession number V00878, from the previously described vector pKCR (Nikaido et al., Nature 311:631-635, 1984)] was inserted into the corresponding sites in the pBluescript plasmid (Stratagene) to generate vector KCR-KS.
A 350 base-pair blunt-ended XbaI-XhoI fragment from the bovine growth hormone polyadenlyation signal ("bGHpA," containing nucleotides 1671-1867 of Entrez accession number AF335419) was removed from the PGKNeo plasmid (Mortensen et al., Mol.
Cell Biol. 12(5):2391-5, 1992) and inserted into the EcoRV site of the KCR-KS
vector to generate the KbpA vector.
To facilitate cloning of the human CAR cDNA insert into the KbpA vector, we introduced an annealed oligonucleotide consisting of restriction sites AvrII, StuI, BglII, EcoRV, and EcoRI downstream of the original EcoRI site and upstream of the bGHpA
site to generate the KbpAlb vector. A XbaI-NotI (gap-filled), human CAR cDNA
("hCAR,"1.2 kilobases, GenBank Accession No. 458541) was then subcloned into the AvrII-EcoRV sites of the KbpAlb vector to obtain the KbpAlb-hCAR construct. A
2.5 kb HindIII-BamHI fragment consisting of KCR, hCAR cDNA, and bGHpA was then ligated into the corresponding sites of a modified bluescript 3'sk vector to yield vector KbpAlb-hCAR3'sk. The modified bluescript 3'sk vector that was used for this step contained unique eight base-pair clusters such as AscI, SwaI, and PacI
cleavage sites that replaced the CIaI-Acc65I region in the 3' end of the original bluescript SK
vector. Lastly, a 2.3 kb fragment containing the albumin promoter digested with NotI and BamHI
(obtained from Ronald Evans, Xie et al., supra) was cloned into the NotI and BamHI
sites of the KbpAlb-hCAR3'sk vector to obtain the Alb-hCAR transgenic construct. This plasmid contains an ampicillin resistance gene and the ColE1 origin of replication.
Example 10: Generation and Characterization of Humanized CAR Mice For the generation of humanized CAR mice, fertilized one-celled embryos collected from C57BL/6 donor females mated with C57BL/6 males were collected in M2 media and microinjected with the linearized Alb-hCAR transgenic construct described above. The resulting mice were tested as described below to determine whether they expressed human CAR. The integration and retention of the transgenic construct in the mice was confirmed by standard Southern blotting analysis. For this analysis, genomic DNA was digested with BamHI and Asp718 and then probed with a 1 kb fragment containing the BamHI-EcoRI restriction enzyme-digested sequence encoding the human CAR ligand binding domain (LBD). The human LBD region was used as the probe because the LBD is the region that is the least homologous to murine CAR. The expected band of about 1.7 kb indicates that a mouse is a transgenic mouse containing the human CAR coding sequence (Figs. 7A and 7C, * denotes lanes identified as containing DNA from a transgenic mouse). Nine transgenic founder mice were identified based on this analysis.
To further confirm that these transgenic mice contained DNA encoding human CAR, PCR analysis was performed. Primers hCAR-hinges' (5'-CCGGAATTCAGGAAAGACATGATACTGTCGGCAGAAGCC-3', SEQ ID NO:
15) and hCAR3' (5'-cgcggatccGGCCGCTGCAGGCGCAGAACTGGTAGGTATGG-3', SEQ 117 NO: 16) were used to specifically amplify the human CAR cDNA sequence and to generate a PCR product of 1000 base pairs (Fig. 7B). As a positive control, primers SCBF (5'-GAT GTG CTC CAG GCT AAA GTT-3', SEQ 117 NO: 17) and SCBR (5'-AGA AAC GGA ATG TTG TGG AGT-3', SEQ ID NO: 18) were used to amplify endogenous mouse (3-actin to produce a PCR product of 600 base pairs. This analysis confirmed that the nine founder mice contained human CAR DNA.
Four of the nine mice lines were also tested by Northern blot analysis to determine if they expressed human CAR mRNA transcripts of the expected size (~1.5 kb). For this analysis, cellular rnRNA was probed with the same probe to the LBD region that was used for the Southern blot analysis. One of the mice lines, number 6210, expressed a human CAR mRNA transcript of the expected size (Fig. 8). The human CAR mRNA, which was under the control of the liver specific albumin promoter, was specifically expressed in the liver of this transgenic line. The other three lines appeared to express mRNA transcripts that were larger than the expected size. These larger mRNA transcripts may indicate rearrangement of the transgene or inappropriately-spliced structures. All of the four lines were analyzed for human CAR expression in at least the following organs: the liver, spleen, small intestine, and pancreas.

Example 11: Generation and Characterization of Humanized CAR Mice Lacking a Functional Endogenous Murine CAR Gene To test the function of the human CAR transgene in mice lacking the endogenous mouse CAR gene, mice from the transgenic line 6210 were first mated with mice homozygous for a mutation that inactivates the murine CAR gene. Male and female offspring from this mating that carried the 6210 transgene and were heterozygous for the murine CAR gene mutation were then mated. Among other genotypes, the offspring from such matings included animals homozygous for the murine CAR gene mutation and carrying the 6210 human CAR transgene, animals homozygous for the murine CAR
gene mutation and lacking the 6210 human CAR transgene, and animals heterozygous for the murine CAR gene mutation and lacking the 6210 human CAR transgene.
As illustrated in Fig. 10, the response of these three groups of mice to two different xenobiotic inducers, phenobarbital (PB) and l, 4-bis-[2-(3, 5,-dichloropyridyloxy)] benzene (TCPOBOP), was examined. Both of these inducers activate murine CAR, resulting in increased expression of the CYP2B 10 gene.
However, it is known that human CAR can be activated by PB, but not TCPOBOP (Tzameli et al., supra). In mice homozygous for the murine CAR gene mutation and lacking the human CAR transgene, no functional CAR is expressed and no response was observed to either inducer. Mice heterozygous for the murine CAR gene mutation and lacking the human CAR transgene express only murine CAR. These murine CAR expressing animals showed induction of CYP2B 10 mRNA in response to both inducers. Mice homozygous for the murine CAR gene mutation and carrying the human CAR transgene express only human CAR in the liver. These human CAR expressing mice showed induction of CYP2B 10 mRNA in response to PB, but did not respond to TCPOBOP.
These results demonstrate that the human CAR receptor maintains its activity and specificity when expressed in the mouse. In particular, the humanized CAR
activates CAR target genes, such as CYP2B 10. Mice expressing human CAR, but not inurine CAR also induces a CAR target gene in response to the human CAR inducer PB, but did not in response to the murine CAR inducer TCPOBOP. These results further demonstrate the utility of mice expressing functional human CAR for the identification of compounds or treatments that specifically affect the activity of that receptor.

Example 12: CAR-Mediated Toxicity in CAR Knockout Mice The effect of PB and the potent CAR agonist TCPOBOP on APAP toxicity was examined in wild-type and CAR knockout mice. Both strains of mice were treated with inducers or the vehicle control, followed by 250mg/kg APAP. For these studies, mice were kept in a pathogen-free animal facility under standard 12 hour light/12 hour dark cycle and fed standard rodent chow and water ad libitum. At least five mice between 8 and 10 weeks old were used in each treatment group. For 3-day PB (100 mg/kg) treatment, mice were injected intraperitoneally three times, one injection per day.
TCPOBOP (3 mg/kg) and/or APAP were injected intraperitoneally. Phenobarbital and TCPOBOP were suspended or dissolved in corn oil, and APAP was dissolved in basic PBS (pH=11). PB and APAP were purchased from Sigma (St Louis, MO); TCPOBOP
was a gift from Dr. Stephen Safe.
For ALT measurements, blood was collected at different times as indicated and transferred to T-MGA tubes (Terumo Medical Corp., Elkton, MD). Serum was isolated by centrifugation at 12,000 x g for 10 minutes in 4°C. ALT activity was determined using Vitros ALT slides (Johnson & Johnson Ortho-Clinical Diagnostics, Rochester, NY) at the Methodist Hospital, Houston, TX.
For histologic examination of the treated mice, the left lobe of-the livers was removed and immediately fixed in 4% formaldehyde-PBS solution, embedded in paraffin, sectioned at 5 urn, and stained with hematoxylin and eosin. Samples were examined under a light microscope. For RNA studies, total RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacture's instruction. Equivalent amount of RNA from 3-5 mice were pooled and 10 ~g of each sample was subjected to northern blot analysis. All cDNA probes were prepared by RT-PCR with mouse liver RNA using Super-Script One Step RT-PCR
system (Invitrogen Life Technologies, Carlsbad, CA). Preparation of probes for CYP2B10 and CYP3Al1 were described as previously (Wei et al., Nature 407:920-923, 2000 and Zelko et al., Mol Cell Biol 21:2838-2846, 2001). PCR primers used were:
CYP1A2: 5'-ccgcctctagagcggtttcttaccaata-3' (SEQ m NO: 19) and 5'-ccgccggatccagccagagtaggcaaatct-3' (SEQ ID NO: 20), CYP2E1: 5'-ggatgaatatgccctacatg-3' (SEQ 1D NO: 21) and 5'-tgatgggcagcaggtctcat-3' (SEQ ID NO: 22), GSTPi: 5'-acagaaactatgtgagcctg-3' (SEQ JD NO: 23) and 5'-atgggacggttcacatgttc-3' (SEQ ID
NO:
24). Hepatic non-protein thiol was measured as an indication of hepatic GSH
content.
Mice were sacrificed two hours after intraperitoneal injection with APAP.
Livers were removed and immediately homogenized in four volumes of 5% trichloroacetic acid and then centrifuged at 1,600 x g for 15 minutes. Supernatant (0.5 ml) was mixed with 1.5 ml O.ImM Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid)] (Sigma, St Louis, MO) and allowed to stand at room temperature for 15 minutes. Absorbance was read at 412 nm using GSH solutions as quantitative.standards.
Based on the above analysis, neither the inducers alone nor the tested dose of APAP induced hepatotoxicity, as indicated by either the serum levels of the liver enzyme alanine amino transferase (ALT) or histologic examination. In contrast, animals treated with either PB or TCPOBOP plus APAP showed strongly elevated ALT levels and hepatic necrosis at 24 hours (Figs. 11A and 11B). The CAR null mice showed no such hepatotoxicity.
Among genes associated with APAP toxicity, PB or TCPOBOP treatment modestly suppressed CYP2E1 mRNA levels, but induced CYP1A2, CYP3A11 and GSTPi mRNAs in the wild-type animals (Fig. 11C). Neither the suppression of nor the induction of the other enzymes was ~ observed in the CAR null mice.
The strong induction of GSTPi expression by PB or TCPOBOP treatments suggests that GSH
depletion could contribute to the xenobiotic induced toxicity, and wild-type mice pretreated with PB or TCPOBOP showed an approximately 50% decrease in hepatic GSH two hours after APAP administration (Fig. 11D). To rule out the possibility that the CAR knockout animals are somehow resistant to NAPQI, the metabolite was directly injected into the livers of both wild-type and CAR null mice, and serum ALT
levels were increased 5 to 10 fold in both. Thus, that both increased NAPQI production and GSH
depletion rnay contribute to xenobiotic induced APAP toxicity.
To determine whether CAR null mice were also resistant to toxic doses of APAP, wild-type and knockout animals were treated with the analgesic at 500 mg/kg, and X00 rng/kg. At either 5 or 24 hours, the CAR knockout animals showed significantly lower serum ALT levels than the wild-type animals (Fig. 12A). This resistance suggests that the CAR null animals may lack a xenobiotic response to the drug itself. To avoid complications associated with the extensive necrosis observed at later times, CYP1A2, CYP3Al l, and GSTPi expression was examined two hours after administration of mglkg APAP. Even at this early time, expression of all three mRNAs was increased in wild-type, but not CAR null mice (Fig. 12B). The resistance of the CAR null animals to APAP toxicity was also demonstrated by the absence of the initial stages of both hepatocellular damage and GSH depletion observed with the wild-type animals (Figs.
15A and 15B). Thus, loss of CAR function results in resistance to APAP
toxicity that is associated with the absence of the induction of APAP metabolizing enzymes.
Since neither APAP nor NAPQI functions as a CAR agonist, this activation may be similar to that of PB, which is based on induction of nuclear translocation, rather than direct ligand binding.
Example 13: Human CAR-Mediated APAP Toxicity To determine whether human CAR is also involved in APAP sensitivity, CAR
humanized animals that are homozygous for the human CAR transgene and the murine CAR mutation were treated with the general CAR activator PB induced expression of CYP1A2 and CYP3Al I mRNAs, and resulted in increased sensitivity to APAP (Fig.
13).
Treatment of the humanized mice with 500 mg/leg of APAP also increased expression of CYPlA2, CYP3Al1 and GSTPi, indicating that human CAR is activated by APAP
(Fig.
16B).
Transactivation by mouse CAR, but not human CAR, can be blocked by the inverse agonist androstanol. Androstanol treatment not only prevents induction, but also decreases basal expression of CAR target genes, including CYP3A11. Androstanol pretreatment decreases APAP toxicity in wild-type mice. To determine whether androstanol administration following APAP treatment has a similar effect, single ' intraperitoneal injections of androstanol (100 mg/kg, dissolved or suspended in corn oil) were given at various times after administration of 500 mg/kg APAP to wild-type or CAR null mice. This 5a-androstan-3a-of (androstanol) was purchased from Steraloids (Newport, RI). For comparison, N-acetylcysteine (1 g/kg, purchased from Sigma and dissolved in PBS) was administered intraperitoneally to other mice. Wild-type mice treated with the inverse agonist one hour after APAP showed a nearly complete absence of hepatotoxicity (Figs. 14A and 14B). This remarkable hepatoprotective effect is mediated by CAR, because androstanol treatment did not block toxicity in identically treated CAR knockout animals (Fig. 14A and 14B). Even at three hours after APAP
treatment, when modest levels of hepatic necrosis are already evident in APAP
treated mice (Fig. 15A), androstanol treatment of the wild-type mice resulted in somewhat lower serum ALT levels relative to untreated wild-type animals. The protective effect of androstanol at one and three hours was very similar to that of the antioxidant N-acetylcysteine, which is used therapeutically to treat APAP overdose (Fig.
17). The serum ALT levels in the CAR (-/-) animals treated with both androstanol and APAP were somewhat higher than those of the CAR nulls treated with APAP alone (Fig.
14A). Since androstanol is reportedly a weak PXR agonist (Moore et al., J Biol Chem 275:15122-15127, 2000), this increase may be due to an activation of PXR that becomes evident in the absence of functional CAR. Treatment of wild-type mice with the strong PXR
agonist 5-pregnen-3b-ol-20-one-16a-carbonitrile (PCN) resulted in an increase in sensitivity to APAP comparable to that observed with TCPOBOP pretreatment.
Relative to the wild-type control, androstanol treatment had no protective effect when administered five hours after APAP treatment.
Thus, CAR is a central mediator of APAP toxicity in mice and humans. CAR is apparently not involved in the toxicity associated with the ethanol dependent induction of CYP2E1 and other targets (Kostrubsky et al., supra and Sinclair et al., supra) since CAR
activation modestly decreases CYP2E1 mRNA levels. However, activation of either mouse or human CAR by appropriate inducers, including APAP itself, results in increased production of APAP metabolizing enzymes and increased toxicity.
Fatal outcomes have been reported for the combination of PB and APAP in humans.
Current therapeutic approaches to APAP toxicity are primarily based on treatments with reducing agents to replenish GSH levels. The results described herein support the use of CAR
inverse agonists as inhibitors of CAR-mediated toxicity in mammals (e.g., humans).
Thus, potent and specific inverse agonists for human CAR provide a clinically.useful means to treat toxicity of APAP or other hepatotoxic agents.

Other Embodiments From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
What is claimed is:

SEQUENCE LISTING
<110> Moore, David Wei, Ping Chua, Steven <120> Screening Systems and Methods for Identifying Modulators of Xenobiotic Metabolism <130> P02729W02 <140> Not Assigned <141> 2003-10-09 <150> PCT/US 01/29672 <151> 2001-09-21 <150> US 10/268,822 <151> 2002-10-10 <150> US 10/219,590 <151> 2002-08-15 <160> 27 <170> PatentIn version 3.1 <210> 1 <211> 358 <212> PRT
<213> Mouse <400> 1 Met Thr Ala Met Leu Thr Leu Glu Thr Met Ala Ser Glu Glu Glu Tyr Gly Pro Arg Asn Cys Val Val Cys Gly Asp Arg Ala Thr Gly Tyr His Phe His Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Val Ser Lys Thr Ile Gly Pro Ile Cys Pro Phe Ala Gly Arg Cys Glu Val Ser Lys Ala Gln Arg Arg His Cys Pro Ala Cys Arg Leu Gln Lys Cys Leu Asn Val Gly Met Arg Lys Asp Met Ile Leu Ser Ala Glu Ala Leu Ala Leu Arg Arg Ala Arg Gln Ala Gln Arg Arg Ala Glu Lys Ala Ser Leu Gln Leu Asn Gln Gln Gln Lys Glu Leu Val G1n Ile Leu Leu Gly Ala His Thr Arg His Val Gly Pro Leu Phe Asp Gln Phe Val Gln Phe Lys Pro Pro Ala Tyr Leu Phe Met His His Arg Pro Phe Gln Pro Arg Gly Pro Val Leu Pro Leu Leu Thr His Phe Ala Asp Ile Asn Thr Phe Met Val Gln Gln Ile Ile Lys Phe Thr Lys Asp Leu Pro Leu Phe Arg Ser Leu Thr Met Glu Asp Gln Ile Ser Leu Leu Lys Gly Ala Ala Val Glu Ile Leu His Ile Ser Leu Asn Thr Thr Phe Cys Leu Gln Thr Glu Asn Phe Phe Cys Gly Pro Leu Cys Tyr Lys Met Glu Asp Ala Val His Ala Gly Phe Gln Tyr Glu Phe Leu Glu Ser Ile Leu His Phe His Lys Asn Leu Lys Gly Leu His Leu Gln Glu Pro Glu Tyr Val Leu Met Ala Ala Thr Ala Leu Phe Ser Pro Asp Arg Pro Gly Val Thr Gln Arg Glu Glu Ile Asp Gln Leu Gln Glu Glu Met Ala Leu Ile Leu Asn Asn His Ile Met Glu Gln Gln Ser Arg Leu Gln Ser Arg Phe Leu Tyr Ala Lys Leu Met Gly Leu Leu Ala Asp Leu Arg Ser Ile Asn Asn Ala Tyr Ser Tyr Glu Leu Gln Arg Leu Glu Glu Leu Ser Ala Met Thr Pro Leu Leu Gly Glu Ile Cys Ser <210> 2 <211> 348 <212> PRT
<213> Human <400> 2 Met Ala Ser Arg Glu Asp Glu Leu Arg Asn Cys Val Val Cys Gly Asp Gln Ala Thr Gly Tyr His Phe Asn Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Val Ser Lys Ser Ile Gly Pro Thr Cys Pro Phe Ala Gly Ser Cys Glu Val Ser Lys Thr Gln Arg Arg His Cys Pr-o Ala Cys Arg Leu Gln Lys Cys Leu Asp Ala Gly Met Arg Lys Asp Met Ile Leu Ser Ala Glu Ala Leu Ala Leu Arg Arg Ala Lys Gln Ala Gln Arg Arg Ala Gln Gln Thr Pro Val Gln Leu Ser Lys Glu Gln Glu Glu 100 105 ~ 110 Leu Ile Arg Thr Leu Leu Gly Ala His Thr Arg His Met Gly Thr Met Phe Glu Gln Phe Val Gln Phe Arg Pro Pro Ala His Leu Phe Ile His His Gln Pro Leu Pro Thr Leu Ala Pro Val Leu Pro Leu Val Thr His Phe Ala Asp Ile Asn Thr Phe Met Val Leu Gln Val Ile Lys Phe Thr Lys Asp Leu Pro Val Phe Arg Ser Leu Pro Ile Glu Asp Gln Ile Ser Leu Leu Lys Gly Ala Ala Val Glu Ile Cys His Ile Val Leu Asn Thr Thr Phe Cys Leu Gln Thr Gln Asn Phe Leu Cys Gly Pro Leu Arg Tyr 210 2l5 220 Thr Ile Glu Asp Gly Ala Arg Val Gly Phe Gln Val Glu Phe Leu Glu Leu Leu Phe His Phe His Gly Thr Leu Arg Lys Leu Gln Leu Gln Glu Pro Glu Tyr Val Leu Leu Ala Ala Met Ala Leu Phe Ser Pro Asp Arg Pro Gly Val Thr Gln Arg Asp Glu Ile Asp Gln Leu Gln Glu Glu Met Ala Leu Thr Leu Gln Ser Tyr Ile Lys Gly Gln Gln Arg Arg Pro Arg Asp Arg Phe Leu Tyr Ala Lys Leu Leu Gly Leu Leu Ala Glu Leu Arg Ser Ile Asn Glu Ala Tyr Gly Tyr Gln Ile Gln His Ile Gln Gly Leu Ser Ala Met Met Pro Leu Leu Gln Glu Ile Cys Ser <210> 3 <211> 42 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 3 gcgcgcgggc cctggcatac attaacacaa acacatacat at 42 <210> 4 <211> 42 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 4 gcgcgctcta gaaggaccca gactctggac ccagggcaaa ga 42 <210> 5 <21l> 42 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 5 gcgcgcgtcg acaggtgaag tgcttctccc caacagaaac as 42 <210> 6 <211> 42 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 6 gcgcgcgcgg ccgctgtcct gggagcagcc tctgcagccg et 42 <21U> 7 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 7 ggacaacctc agcccacagt gatgc . 25 <210> 8 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 8 tcctttggtt accacctgac tctgc 25 <210> 9 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 9 ccgcctctag aagtcaacat tggttagac 29 <210> 10 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 10 ccgccggatc ccacactaag cctcataat 29 <210>

<211>

<212>
DNA

<213> e Mous <400>

accaggaccatggagcccagtgtgctgctcctccttgctctccttgtgggcttcttgcta60 ctcttagccaggggacacccaaagtcccgtggcaacttcccaccaggaccccgtcccctg120 cccctcttggggaacctcttgcagatggacagaggaggcctcctcaagtctttaattcag180 cttcgagaaaaatatggcgatgtgttcacagtgcacctgggaccaaggcctgtggttatg240 ctgtgtggaacagacaccataagggaggctctggtgggccaagccgaggctttctctggc300 cgggggacagttgctgtcgttgagccaaccttcaaggaatatggtgtgatctttgccaat360 ggggaacgttggaagacccttcgtagattctctctggccaccatgagagactttgggatg420 ggaaagaggagtgtggaggagcggattcaggaggaagcccaatgtttagtggaggaactg480 cggaaatcccagggagcccccctggaccccacgttcctcttccagtgcatcacggccaat540 gttatctgctccattgtgtttggagagcgctttgagtacacagaccgtcagttcttgcgc600 ctgctggagctgttctatcagaccttttcactcataagctcattctccag.ccagatgttt660 gagctcttctctggcttcctgaagtactttcctggtgcccacagacaaatctccaaaaac720 ctgcaggaactcctcgactacattggccatagtgtggagaggcacaaggccaccttggac780 cccagtgttccacgagacttcattgatatttaccttctgcgcatggagaaggagaagtcc840 aaccagaacgcagagttccatcaccagaacctcatgatgtctgtgctctctctcttcttt900 gtcggcaccgagaccagcagcaccacgctccactatggcttcctgctcatgctcaagtac960 ccccatgttacagagaaagtccaaaaggagattgatcaggtgatcggctcacaccggcta1020 ccaacccttgatgaccgcaccaaaatgccatactcagatgcagtcatccacgagattcag1080 agattttcagatcttatacctattggagtgccacacagagtcaccaaagataccctgttc1140 cgagggtacctgctccccaagaacactgaggtgtaccccatcctgagttcagctctacat1200 gatccacagtactttgaacaaccagacagtttcaatcctgaccagttcctggatgccaat1260 ggggcactgaagaaaagtgaagcttttctgcccttctcaacaggacaaatttttgatcaa1320 aagtctgtgg gaaagcgcat ttgtcttggt gaaagcattg cccgcagcga attgttcctt 1380 ttcttcacgt ccatcctcca gaacttctct gtggcaagcc atgttgctcc taaggacatt 1440 gacctcactc ccaaggagag tggtattgga aaaatacctc caacgtacca gatctgcttc 1500 ttggcccgct gattgggctg aggcagacag gggtcaccag taatgttgag aatgactctg 1560 tctttgagcc tctgagacag ctggtggaaa tcagtactcc tattgcatgt ctccaaatct 1620 ccagggctcc aaggcatgtt cttcttccct gtgaatggca ctggagaaat caatcaactg 1680 tctttcttga catgtgaaaa gagacttctg gagtccacat ctcatgttga gtcacttccc 1740 ttttcctccc aatagcccaa gtgtccactt atcagctccg catgatctgg gatctgtgct 1800 aatggactct gtataaggtc tg 1822 <210> 12 <211> 42547 <212> DNA

<213> Human <400> 12 gatccaggggtctgccgtagatcttagccatggactgcacctggggctccgtttgaagaa60 ctatttgtagttttacagcttcaattctggaagagacaaacttaacaaggaggttaaaga120 cacagggattgaaatgtctggcctgaagtgcaggggattatttctttggcacacttcaca180 ~ggccctgactacctgcttgatagttttgaaaaggcctggtccagcaaataatgatttggc240 cattggatgggtgctatcaacgcctaaatgaaaggtttggtgaagggttt.taagtaattt300 ccattggttagctgcaggcaaaagtatttttccttctttggtggctagacatcctgaggg360 gagggaactatgtccttgtgaggtaccccattctatttcttctgctgagcactggggctt420 ggtttcctggaggggattaccccatactagggttccttctataagcatttctaatggagg480 gtcccgccttgtggctctttggcttcaatatctgcttggtggttcccttctatttccctt540 tcctttcctttctgatgaccccagcagtgtaagatggccacctctttaggtttccgtaca600 gcccataataactcataatggcttcctgatgtttaataggtgttccctcagaagttagga660 attccatttctcaccatatttttgtgtaggcatggaggagaaggtaagcataattagagt720 gtttatatatatttacccttttcccttctcctaattctagtgtatcatggcccctgcttt780 tgctaggatgtctctccctaacaaaggagtggggctttcaggcataattagaaagg.catg840 tgaaaagagtaaagtcccccagttacaacttagtggctgggcgaagtaactagtgactgc900 ttgtcctaggacccctcggatagtaacagatctggaaatccagctagtcctgtctctcaa960 aactacaatgataaacaataagagaaaaagaaaggaagaaaggatatacatacataaaga1020 agccaactaataatatgacaggattaagctctcacatatcaataataaccttgaattaaa1080 tggattaaac tttccactta aaagaaagag actggctgaa tggatttaaa aagcatgacc 1140 cagctatatg ctgcctacaa gaaacacatc tcaccaggaa agacatatat agtgaaagta 1200 aagaaatgag aaaagatatt ccatgcaaat aaaaaccaaa agtgagcagc aatacctatg 1260 tttataaagg aaacagactt taagtcaaaa actgtaaaaa gagacaaaga aggtcaatac 1320 ataatgataa aaggatcaat cgagcaaagg atccatgagg aaattcctgc ctaataaatt 1380 ttggtcagac cggttgtctg ctctcaaacc ttgtctcctg ataagatgtt atcaatgaca 1440 atgcttgccc gaaacttcat tgcaatttta atttcacccc ggtcctgtgg tcctgtgatc 1500 tcgccctgcc tccatttgcc ttgttatatc ttattacctt gtgaagcatg tgatctctgt 1560 gacctacacc ctattcgtac actccctccc cttttgaaaa tcactaataa aaactttctg 1620 gttttacggc tcagggggca tcatggaacc tgccgacatg tgatgtctcc cccggacccc 1680 cagctttaaa atttctctct tttgtgctct gtccctttat ttctcaggct ggccgacact 1740 tagggagaac agaaaagaac ctacgtggaa tattgggggt gaattttgcc cgatatctgg 1800 ctgaatttcc cctgataatg ccactctcta tgtccatgtg. tacacattgt ttagcaccca 1860 cttatgaatg agaacatgtg atattcactt tctgtgcctg gcttgtttca cttaagataa 1920 tcccctccag ttgtatccat gttgctataa aagacattat tttattcctt tttatggcta 1980 aatagtattc aatggtg.tat atataccaca ttttatttaa ccattcatct gttgattccc 2040 tatttttgct attgtaaata gcatttggac cacatttcaa gtacttaatt agtggccaca 2100 tgcagcaagt gactatcaca ttggacaatg tagccccaac ccactgtatg accttgggta 2160 agacttgcaa actgtcattg cttcaatatc tccatctata aaatggggat gg.caacaata 2220.
cctcactaag agtgtaaaga ctgagttact gtgtgtaaag cacttcacgc ctccccatcg 2280 gtgcttcacc ctggggctgc aatgagcacc caatcttag-t gtcagatgac acagcacagc 2340 aagacegagg cccttggttc aggaaagtcc atgctgccac ctcttcaggg tcaggaaagt 2400 acagtttcca cctcttacaa ataggactgt ttgtctgctc ctcctgggtc aaagtaactt 2460 cgggttcagg tcctggatcc agcaaagggt ttgcttaaca ttgcaagaaa gatgttgcct 2520 catggtcaaa agtcaggcgt aggatgagac aggcagacac gcacacattc acacccacgt 2580 tttgcaaaga tggactgacc ctgtcagagg atgtgtgggt gaaggtgcac agtgaggata 2640 gagacatatg ggagtccagt agacatcaat caaactggac tcagtttgca cacacctgga 2700 gctcaagagt ctccaggggg aaaacagaga cacaaagtca gacagagaga gagccagaga 2760 aatttcctgc accgtgaaga tagtcagagg cagggaagaa actccttagc actagttaga 2820 gtgatcagaa accaagagga cctgatcgct gtacctgcca ggtctcagtt tctgtctcct 2880 tccaactgac cacctcttcc tctgagactc accagttctg catctcttgc tcctccttct 2940 gtttctccga ccacttccac ctgtggctgt cacagaaggg cggatgaagg aggggacact 3000 ggagatagac tcagcatctg caggcttcca aagagagggg ctaggagatc caccaacaca 3060 ccagcacaaa tacaccagca cacacagata cacacaattg gttcatgtat tgctaggtta 3120 cagtttgcta tgctacaaag gcagtaggcc aaatttgatt gaattgaata attccttatt 3180 ttcatcagct tctccttttt tttttttttt tttttttttt gagatggagt attgctgtgt 3240 cacccaggct ggagtgcagt ggtgtaatct tggctcactg cagcctccac ctcccaggtt 3300 caagtgattc tcttgcctca gcctcccgag tagctgggat taaaagtacc caccatcacg 3360 cccggttaat ttttgtgttt ttagtacaga tggggttttg ccatgtgggc caggatggtc 3420 tcgaactctt gacctcaatt gatctgcccc cctcagcttc ccaacgtgct gggattacag 3480 gtgtgagcca ccgcacccag ccagcctctc agttttgaac atgcactacc accacctcca 3540 caacacacaa atgtaaatgc actttcgtat ataaaactgt ataaatacaa ggaagctcat 3600 acacatgcaa ggatacacac ataagcaccc ccagattcaa ccacagaaat atacgccagt 3660 acatttgcat aaattcaaac acccctttac atgtaaaaat catataagca catacaggga 3720 tgcaagcagg catggacaaa tgcatgcaag cacagacaaa cagacaaagc taagtaaaaa 3780 agtgcaagct cacctatgct tacaaaaata gacatacata tacccacaaa cccacacacc 3840 cacacattca cttgctcacc tggactttga tatctctacc actgtatccc tgccaatatc 3900 tacagagtgg gtaaagggat aggcatcagg tcactgggtt gcccaag.cag gaagtctggg 3960 ttccctaaca actttttcta agctaatgct cctggatgat gatgaaaaag gaggtgggga 4020 atggatgaaa ttttataaca gggtgcagag gcagggtcag gataaaaggc ccagttggag 4080 gctgcagcag ggtgcagggc agtcagacca ggaccatgga actcagcgtc ctcctcttcc 4140 ttgcactcct cacaggactc ttgctactcc tggttcagcg ccaccctaac acccatgacc 4200 gcctcccacc agggccccgc cctctgcccc ttttgggaaa ccttctgcag atggatagaa 4260 gaggcctact caaatccttt ctgagggtaa gacacagacg aatggggtct gagggtgagc 4320 tgcttcttgc cttggtactt ggggaagctt caccaaacag aatgaggcag acttccagag 4380 tcaggggtgg cacgggcatg gttggtgagt acggagcatg gtgaagcatg. atgggtggta 4440 ttattaggag aaaagcatca aattaaattt agcagagttt atttgagcaa agaagtgact 4500 catgagttgg acagctccct aaaccaggaa agacaccaca cggcagtatg. gtcaagtggt 4560 atttacaggc agaaaaagga ggtgacatac agaaacagcc tgattggcca cagatcacag 462-0 cttgccttac ttggtcacaa tctgagcagt ttgcagcctg tgtggactga aagcccagct 4680 gctctgatta gccaacactt ggctacttgt cacaagaata tattcatttg ggccaggtgc 4740 agtggctcatgcctgtaatcccagtgcttttggaggccgaggtggtggatcacctgaggt4800 caggagttcgagaccagcctggccaacatggtgaaaccttgtctctactaaaaatacaaa4860 tattagctgggcatagtgatgcgtgcctgtaatctcagctacccaggaggctgaggaagg4920 agaatcacttgaatccaggaggcagaggttgcagtgagccaaaatcttaccactgcactc4980 catcctgggtgacagagtgagactccttctcaaaaaaaaaaaaaaaaaaaagaatatact5040 cccaagttaggttgcagttcactctacagagagagctttaggtcaaatttaatttaatta5100 aacaattctccccttttggtcagcctcaaaattttgagattgaccaaaaccttgggcatc5160 aacattacttctgtcaccatcataatggacttgtctgctctcagtatggaattcacaatg5220 gacaatgtcaacgtagttgagtgattctttaccttttcttcatgtttttgttgttcccac5280 tgtaatgagcccactggatgtacaaagaatggctgcatatgagcatttaagactcttttt5340 ttttctgagacagggcctcactctgtcagccaggctgaagtgctgtggcatgatcacgtc5400 tcactgcagccttgacctcccaaggctcaagtgatcctcctgcctcagccccccaagtag5460 ctggaactacaggtgcatgccaccacgcccagctaatttttgtattttttgtagagacag5520 ggttttgccatgttgcccagactggtcttaaactcctgggctcaagcaatccacctgcct5580 cggcctcccaaagtgctaggattacatgtgtgagccaccgcacccggccaagactcttga5640 gaaaatacaacacatcagggagactgttatgatggctctcaggagggtaatacgaagaaa5700 atgaagtcactgggcctgtaataaactttgaggaatg.tgg,acttgggggtatagataagg5760 tccactgtccacagagagaagaaaggctgttaatagtctcttttaacttgagtg~tgtcca5820 tgaaccaaactgatcaaaatcgaataattcgaagttcagacaataaag.atagttcaatag5880 tattagagtccaattggtcatagattttgttcagggcatgatggtaattaaggaccagag5940 cttgctataaaataacttgatttatagagacattcatttgtagttggcctggtaacatat6000 agtatcctggagacccactagaagaaacattaagagtagaaaagtttgggatagccaggc6060 ttgctgtgttagtccattctcacactgttataaagacatacctgagactgggtaatttat6120 aaacaaaagggatgtaactgactcacagttccacatggctggggaggccccaggaaaata6180 caattcatggcaaaaggtgaatgagaagcaggaaacttacaatcatgatggaaggtgaag6240 gagaagcaagtaccttcttcacaaggtggcaggaaaaagagagagagccaagggggaaga6300 gcctcttataaaaccatcagatcttgtgagaactcactcactatcacaagaacagcatgg..6360 gggaaaccgcccccaggatccagttacctcctactaggtccttccctccacacctgggga6420 ttacaattcaagatgagatttgggtgaggacacagagccaaaccatatcacttaccatca6480 ccattcaggatgcttgcaaaccaactgctagctgcacctgtaaacacatatctgtttctt6540 tcccctgaga aatgtcctta gtgtatttgt ggcagtgtct agagaaacag cagtgtcagc 6600 cgcattttaa attaagttat ctgcactagt gaattcactg gaaagataag agcaatattt 6660 ggttttcttc agcaccatac acaagcctcc aagatgggca tagaggagat ctaaaattgc 6720 gtgatgttcc attaagcgtt tttgttgcca caaatgttct catctcagtt tggagagtgg 6780 cttctaccca tctgaactcc ttggaggttc aattagctgc aaaattcaag atgtccctta 6840 atgtataact tagcctcaga ttccatacaa ctgtcaccca aataccacca agaatgagca 6900 cccaggaacc caactggaac cttttctgaa cagaaaccaa cttatcttcg tcgattttga 6960 ggttgatagt aatttcagtt attgactgtt ttggctttta actatgggag gtattaggaa 7020 actctcaggg aaacaatttg gaaagcagca gtgagctagg ccaaatagca agttctggac 7080 ctgtgaggag aaagaacaga gtaagcaaac ctcaagatac tcaaggtagg cactcgtggt 7140 gttggaaaag agggtcacct actggcatta gagcagagat cagttagatt tgtttaccca 7200 taagtctgca tagctcctga acaaggtggg aaacttactt ttttgtggtc tttttctagc 7260 atgctgcgaa ggtgcataac cacatttagt tggaaagaga ctttactgta tttacttatt 7320 tatttgtttt taatagagtt ggggtcgtgc tatgttgaac tcttggcctc aagcaatcct 7380 cccatctcaa tctcccaaag tgctgggatt acaagcatga gccaccatgc ctggccactt 7440 tacatattta atccagtaac attacacacg caattgccca cacccccata ggtagtcccc 7~500 aggtcttgca tacgggatgc ctggaagcaa aatatgcctt ttgcagccat tattcag.ata 7560 catttcctat atttagtagt gattatgtta ttagctagtt aatagtatgt tattacgtac 762.0 tg.ttattata ttaactaact aataacataa tcactactaa atatttccag tgagtgcaaa 7680 aaagcaagtg gcaatgatgt ctagaatatc aagatatagc tttccactcc tcctttggg.g 7740 tttctgggtg attctcattg ggaacatgaa gaggcattgg caccagtgaa attatttcct 7800 gattttgggg cgttggttca gaaactcaac aactccttgt tttttttgtt tgtttgcttg ,7860 ttttgctaga gtataagcct ttgctaaagc cattcacaga ttatagtcct atgg.attttc 7920 ttgtaaggaa agggaaaggg ttaggacagc aagaaatggg gaagaaagga taaaagataa 7980 tgctttcatg atggaagaga aatcttgatc cacaatcttg gaaaagctgt ccgcatataa 8040-gatgccaact gcttctgggg aaaaacttcc ctggtcagct ttgccttaag gtctccaaca 8100 gacatacagt tctaggagtc tagaagggtc ctttccaatg gagagatgtg gatccaagat 8160 ccgagaccct gacattttgc tacagagaag aacttggcat tgtccttccc aatggagtac 8220 aaggaacagt cttagaagaa ettggtacgg tctcttccaa tggagttcaa ggacagtttg 8280 tctggtgtca tttccaaagg gcccaacctc taaattctag atcatgaaag gtctggttgt 8340 catcaaccga tgtgtcatca atgactcatt ttacctggtg aaaacatgct ttggcataaa 8400 gtattatagc cttgcattat tgagtcatat cagagtttat aagagtggga gatacatgag 8460 attctattat taggggcata ggccctctat tactatttta caagagatct atctatgtct 8520 ttccagtagg agtggatctg attgccatca atcaataata cctgagacca agggactcca 8580 atcaattcag catgctttgc ctaatgatat ttgtttgtaa tactgttgcg ggacaatcaa 8640 agactggaga gaccaaaaaa ggttcaggag agtttattaa attaaggtga tcaccggttc 8700 agccagacat acatccagaa agtctgagcc ccgaacaaag gcttttccta cttttaaaca 8760 tattaaggtg ggaactacat gaggcaggaa gccagtttca gaagtgagaa acaaagcagt 8820 taaataacat ttcttacatc ttgagaaaga catgtcttgc aacctaacct tatcggtccg 8880 gtgaccctgc agctgtgcag gaactcactg ggcctgtaat aaactttgag ggatgtggag 8940 ttggggagta taggtaaggt ccactgtcca cagagagaag acaggctgtt aatattctct 9000 tttaacttga atgtaaggtg tggtcatact ttgcagcaac cttaagagga ttttaaaatt 9060 tatattacta ctactattag gttatagttg atttcattaa ttccttcttc aataccttat 9120 ttaactgttt taccacttgt ctagtgaaac aagtacctct gtcactggag agttctccag 9180 gaatgcccag taaggaaata cattttctaa taacctttta tctacggtta tggcattgat 9240 tgatctttgt gcatagaaat gctttaatac aaccagaaaa catgcaatga agctggcagt 9300 tgaattaact ccagcttcaa gtgttcaaat gatctaccaa gtgccagaaa tatatcacct 9360 gaggtttttg ttgtcttact agaattatgg atttgataaa ccaaatatta gttataaacc 9420 atttagtaat cttagaatag tcacctcatc aatatttttt cactgtttgg atcattttct 9480 ctcttctatg atgagtcatg gaatacggag cttttagtaa tggaaatttt aagaactcag 9540 gaaggaccag gcggccatct agggtctcca tgagtgcatg cttcacattg gaattacaga 9604 ctctaaagta caaattttaa tacaatgagt tgcaatttat gcttctctaa ttcaggtaca 9660 taacactggt ttattaaata ggttatcata ggtaatttga tggtgccatt gcactccagc 9720 ctgggtaaca gattgagacc ctgtctctga attggaactg cacgggggca ctgtccttgg 9780 aggggtgaat gggcatgaag aggtgtctgg gtatgagcca caggtataga atttcactct 9840 tctctgccat cctctgttac atcctgggta cctgcctgcc actgaaagaa tgaggtaaaa 9900 gaggtggtgg cacgaatcaa atagatcttg ctgtgccaat gagagagagc agactagccc 9960 atgtcagtgc caggagagtg gaggagagag ggagagcagg agaggagtgt gggtagggag 10020 tgctcatcaa cagtacacat agtgccctat accggtaact gccactggct cagtatttac 10080 ctgggttatc actgcttacc atgcctgatt ttatgattaa ttatctactt atcattacta 10140 atccatcaac ccactttcca atgggagaat tagaacactg acaatacctt ccagctcctc 10200 ttccccttcc ccctcccatg ttgaccatac tcctgaatct taggctcgtt atccttttac 10260 tattaataga gttatttttt taatttctga tcaatggtct ttttaatgat accaagtaca 10320 gagtatatat gccaatacta ctatgaattt ttaaattatt tgctcagaca gaatacatgg 10380 acatacaaat gatgaatgtg ataattgtca tacatatata tttatcttag acacttagtc 10440 aaaacatgtg gtctttggtt atcagttaga cactgtcact ttacctggaa gatacaaggt 10500 aattgg,tcac ag.actctcaa attatgaaac atttagattt ttcaggggaa tgacatggag 10560 ggagccaagg agtcttatga ttagataaga tgttgtttgg gtggctcacg acaccactgg 10620 gcaacactca aagaggtggt ggcttatacc tgtaatccca acactttggg aggctgaggt 10680 gggaggatcg cttgaagcca ggagttagaa accagcctgg gcaaccaaac aagacctggc 10740 tctacaaaaa agttttaaaa atttagccag gcatggtggc atgtgcctgt agtcccagct 10800 acttgggaga ctgaggcagg aggatgactt gagccttgta gtttgaggct gcagtgagct 10860 atgatcacgt ca~ctgccctc cagcctgggc acagagcaag accctgtctc ttaaaaaaaa 10920 atcatctgca atgtgaggag tgataacatt taggaacgtg tgtatagg-tt taaatgctgg 10980 tcaaagacat cctacacaat tcgctgaacc ttctctctaa gggttttttc ccaagctctg 11040 cagacgctat ctgggcacaa atcatgcctc tgttaacaga atttgctgtt ccttctagct 11100 cttggtatcc cactgcccgc tttctttatg aaagctggta tggtcattga atatcccaat 11160 cctttacaaa tttggaaaca agcaaaactg tcaatgaaat ttgtatttgc ctaaaatgag 11220 ttttctttct ttctttttta ttattattat actttaagtt ttagggtaca tgtgcacatt 11280 gtgcaggtta gttacatacg tatacatgtg ccatgctggt gcgctgcacc cactaactcg 11340 tcatctagca ttaggtatat ctcccaatgc tatCCCtCCC CCCtCCCCCC aCCCCaCaaC 11400 agtccccaga gtgtgatgtt tcccttcctg tgtccatgtg atctcattgt tcaattccca 11460 cctatgagtg agaatatgtg gtgtttggtt tttttgttct tgcgatagtt tactgagaat 11520 gatgatttcc aatttcatcc atgtccctac aaaggacatg aactcatcat tttttatggc 11580 tgcatagtat tccatggtgt atatgtgcca cattttctta atccagtcta tcattgttgg 11640 acatttgggt tggttccaag tctttgctat tgtgaataat gccgcaataa acatacatgt 11?00 gcatgtgtct ttatagcagc atgatttata gtcctttggg tatataccca gtaatgggat 11760 ggctgggtca aatggtattt ctagttctag atccctgagg aatcgccaca ctgacttcca 11820 caatggttga actagtttac agtcccacca acagtgtaaa agtgttccta tttctccaca 11880 tcttctccag cacctgttgt ttcctgactt tttaatgatt gccattctaa ctggtgtgag 11940 atggtatctc attgtggttt tgatttgcat ttctctgatg gccattgatg ttgagcattt 12000 tttcatgtgt tttttggctg cataaatgtc ttcttttgaa aagtgtctgt tcatgtcttt 12060 cgcccacttt ttgatggggt tgtttttttc ttgtaaattt gtttgagttc attgtagatt 12120 ctggatatta gccctttgtc agatgagtag gttgcgaaaa ttttctccca ttttgtaggt 12180 tgcctgttca ctctgatggt agtttctttt gctgtgcaga agctctttag tttaattaga 12240 tcccatttgt caattttgtc ttttgttgcc attggttttg gttttttaga catgaagtcc 12300 ttgcccatgc ctatgtcctg aatggtaatg cctaggtttt cttctagggt ttttatggtt 12360 ttaggtctaa cgtttaagtc tttaatccat cttgaattga ttttgtatac taaaatgagt 12420 tttcaaaagg atctttgtgg ctaccttatt agttcataga aagtggaggc ttgtctggaa 12480 tgatattaaa gaattttttt cattttaatt gttttagaga cagggtctca gtctgttacc 12540 caggctggag tacaatggca cagtcatagc tcactgcagc cttgaactcc tgtgetcagg 12600 agattctccc accttagcct ccagagtagc tgggactaaa agtgtgagcc accatgcccc 12660 actatttatt tttgtagaga tgtgtttggg ggacggtctc actatgttgc ctaggctggt 12720 ctcgaactcc tggactcaag caatcctcct gcctcaacct cccaaagcat tgggataagt 12780 tttgcataga catgtttgac cctctccctt cctttattgc agcaaagatt tccattttct 12840 cacagggagg acttggggca aattgttttt gtcgttgtta ttgttatttg aggatgtggg 12900 tgggtcatct tggttaatgt cagtgcaaga acggtgtctg ctcctcatta ttggggttcc 12960 ttagctattc aggcaggcag cagctccctt gagaagactt cctgaccccc aggtccccat 13020 catatgctct catacaccag ctgcccctcc ttactgagcc tatgtccttg atagtgctgc 13080 atttacctgt gatcctgtgg. ctgagctctc cctcacactt ccagacaagg aaggcctgtg 13140 aggtatcagc acagtgctga acatagtacc tggtacacaa taggcattta gtaaatatgt 13200 gaacaggaac aaatgaagga gtcagtgag~t gaaagctcca agcctgactc agcggaactg 13260 gcagtcggcc agggcctaca aagtgctgcc tggccctcag tagggggtgg cagatctggg 13320 gatccctctt caccaaatag. agttgcatca tacaggtaaa ggtcccaagt gcctattgtt 13380 ttcttttcat cattttccat gtgtgacaag agtatcacgc aatcatgtga atcaatggac 13440 ttagttttct cactagacta atgtgtctag gaattatctg tgttgtcatg gggattttcc 13500 aggtgtcatt tacaactacc tgtggacaag atgaggtgct accctcatct tagaattagg 13560 gatggtggct gggcatggca gcccatgcct ataatctcag cactttggga ggctgaagca 13620 ggtggatcaa ctgaggtcgg gagttcgaga ccaacctgac caacatggag aaaccctgtc 13680 tgtactaaaa aacacaaaac tagctgggca tggtggcaca tgtctgtaat cccagctact 13740 cgggaggctg aggcaggaga attgcttgaa cccaggaggc ggagtttgcc ctgagctgag 13800 attgcatcat tgcactccat catgggcaac aaaagtgaaa ctccatctca aaaaaaaccc 13860 aaacagaaac aaacaaacaa acaaaattag tgatagtgat gctcagcctg gggaaagcat 13920 ttgtccagtg gcacacaact gggaaaaggg gaagctgaga tccagcagga ggtctgtctc 13980 caaagccctc ctagactaaa gctgcttaac aatttgtgga ttatgaaatt ctcatagagt 14040 ttgattaaag ctgtggcccc ctcttttcca aataggcaca tacatgtttg cataaagttg 14100 tgaggcttca cacactccct aaaactcttt cataaacctt ccaaggatcc tctaggtatt 14160 cacgaccatc tattatgatt gcatctcttg ggggtggggt aaagagggag ggcatgagca 14220 agtgtgcatc agggctgagg aaggtggcgc tgttgcttct ccatttccca ataagcttcc 14280 agattctttt tgatgtcaga gggatgtggg ctcgtgtctc agattcaacc cgtatgcatt 14340 gagtcacagt tttctcttcc gtcagttaag atcatgacaa tgagaatgtg tgcccctaag 14400 gtggttgtga ggattaaatg ggatattgca tacagctagt atataataag tgctcattaa 14460 atggcaacta ccttgatcca ctcattcatt tattcacgaa tccaataaca attcactggg 14520 cactttctat gtaccaggaa atagtctagg aattgatgat gtggcatctt ggacaagaca 14580 tacgaggtca ctgcgcttat ggacattcca ttggcagaga cagataagca aaaaataaac 1464U
agataaggaa atgttaggtg, gagaagagct acaattaaac taaagcaggg ggatatttca 14700 gacagtgatg agaactacgt tagattgagt ggttagggga agactgtctt gttttttttt 14760 ctgagcagag acaatgaaag attcaggtgg aaggcactag aaggaggcag gggtggctac 14820 aagaatcctg aaacaggaaa aatg.tcagag aacaacatgg aggaatgagt gggaggagaa 14880 gtcagagcgg taatagggcc acgtcatgta ggaccatgtg aaccctcagt ggagacttca 14940 gatcttattc tcagcaaagt gggaatattg taggccttgg tgagcacgtg gatattaatt 15000 attttacatt ttagtggggt taccctggct tgtggggcag tgaaacaatt gttaagtgga 15060 gtaagcatgt catccagaag tccagtgaag gggctttgca gagatcaatg~ cagggcattt 15120 gtaagtaaac ttaacaaatt ttatgacttt tatgtcaagt ttttattgaa cttgaacatc 15180 actgtaaaaa gcgcacagct caatgaattt tcacaaactg aacatactca tgtaattgac 15240 atgcaggcca agtagcaaga atgccccgaa accctgcacc ctgttctttt ctattcacta 15300 tccacctacc atcttgatca tgggttgatt ttatctgtgt atgaacttct tttttgtttt 15360 tgtttgtttg ttcctgagat tgaattttgc tctgtcgccc aggctggagt gcagtggcac 15420 tatctcgcct cactgcaacc tccgcctccc cgggctcaag caattctctc gtctcagcct 15480 cccgagtagc tgggactaca ggtgagagcc accatgcctg gctaattttt gtatttttag 15540 tagagatggg ttttcaccat gttggtcagg ctggactcaa actcctgacc tcgtgatcag 15600 cctgcctcag cctcccaaat tgctgggatt acagatgtga gtcaccacgc ctggccctgt 15660 gtttgaactt catacacata aaattatgat gcattgactc ttttgtgcct agttgcttcc 15720 actcaacatt atgcctgtga gtttcagcca cgttgtcgcc tgtagctgtg gtttgttctt 15780 ttttgttgct gtatactagt ctattgtgag aaccctcact gattgtctat atctatattg 15840 atgagcattt tattaagaaa gcttctgtga atattttgtt cattttttgg tgaacacaca 15900 tacacatttt tgctgggcat gtacctggga gtgaagtggc tggtttcctg ggtatccatt 15960 tgttcagctt caggaaatac tgcccagcag ttccccaagt ggctgcacag taatcccacc 16020 ttatccactg gagatatctt ctgagacccc cagtggatgc ctgaaacccc acataatact 16080 gagcactatg catactcttt ttttttccta tacaatcaca ttatataggg agggtggtat 16140 acacagtgca gacatggtgg acaaaggatg attcgtgtcc ggggtgggac agagtggatg 16200 gtgagagata tcatcatcct actcagaatg atgcacaact taaaacttac gaattcttta 16260 tttctggaat tttccattta atattttcag actgcgatta gctgcaggta actgaaactg 16320 caaaaagcaa aaccacagat cataagaagt atgcggtggg. ggtgatattc atcgtatttt 16380 atcaaccatc tttactgttt agggcacaag ccaatcagag cagaccctgg ctgggccacc 16440 cattaaccta agcttgtcca acctgcctta ttttgttgtt gttctg~tttt gttttgtttt 16500 agctttttag cagcctgaag ccatggtttt cagtttctgt ctccagtgat acacagaaag 16560 gaaggatgag gaaggggctt tactggccca accagaaacg gaaactaaga~ acccatgact 16620 gtattctctc ccttggacag cgttaaccat taacccttaa ttgctgggtc ,ccagcagggg 16680 aaagggcagc ctggggaggc ggatgttggg gaggggctaa ttaccaatct .ggtatgtaag 16740 tattttgata gttttacaaa tgaggtgtat gctgactaac agccacccct ggtgtggatg 16800 tgattggcag ttccgagaga aatatgggga cgtcttcacg gtacacctgg gaccgaggcc 16860 cgtggtcatg ctgtgtggag tagaggccat acgggaggcc cttgtggaca aggctgaggc 16920 cttctctggc cggggaaaaa tcgccatggt cgacccattc ttccggggat atggtgagag 16980 cctcagaggc actgggaggg ggcgggtggg gggtgcatca gggaagggag tatatgggag 170~40 gaagaaggac tcagagcctt cttccaactt cttctacaac caacccacac ctcccctgca 17100 ccccaggtgt gatctttgcc aatggaaacc gctggaaggt gcttcggcga ttctctgtga 17160 ccactatgag ggacttcggg atgggaaagc ggagtgtgga ggagcggatt caggaggagg 17220 ctcagtgtct gatagaggag cttcggaaat ccaagggtga gtcctggggg atgaatagga 17280 aagaaagaca atgaaacact gagagatgca ggtgcacggg aatagaaaga cagagaggta 17340 tataagggca cagacagaga cagacgaaac tggagacacc atcagacaga gggatagaga 17400 cagagaggga gagagacagg ggaatagaga gggatgggga tgggcaggag agaaacacag 17460 agagccaggg aaagagagag atgccaggtg tataatgtcc aagagttact caaagaggct 17520 ggatgtgatg actctcacct gtaatcccag tactttggga agctcaggca ggaggattgc 17580 ttgaggccaa gagttggaga acagcctggg caacataatg agatcctgtc tctacacaat 17640 atagaaaaga agtgagccac gcatggtggt gtgtgcctgt agtcccagct actcaggagg- 17700 ctaaggtggg actacaggat cacttgagcc caggaggttg aggttgcagt gagctgtgat 17760 tgtaccactg cactccagcc tggacaacag agcaagatcc tgtctcaaac aaacaaacaa 17820 accctcaaag acatataatt tcatggatca attgtgtctg tcaaagtcaa aaacggaagt 17880 taagtaaaag aaaaaaacta cagacattta acaaataatg aactgtgttt tccttgccct 17940 gggtgaagtg ctgatg.agct ggcagtgagc agacaggcca ggtggggtgt tctgcccg.gg 18000 tgcagctgga ggggtcatca aagaatcact aggttatttt tgagttctcc ataacttggt 18060 gtctgtgaga catgtaggtg aagggtctct ggctagcacc tccatctcat tcatgccagg 18120 tgtttaccat ctctcttatc aaaatttctc aagagactct gggatgtaaa tgcagaggct 28180 gcatggggag gtagagaccc aggagctata gggaaacggg- gacaagaaga ctgaagagaa~ 18240 ggacaggaag aaacagtgac acaggcagga ggaaagagac agatggaggg accaaaacaa 18300 aaaaatatag gttgggtatg gggctcatgc ctgtaatccc agcactttgg gaggctgagg 18360 ctggcggatc atttgaggcc agcagttcaa aaccagcctg gccaacatgg tgaaacccca 18420 tctgtgctaa aaatacaaaa attagccagg cttggtagca cgtgccttta atcccagcta 18480 ctcaggaggc tgagacagga gaattgattg agcctgggaa atggaggttg ccgtgagcta 18540 agatcacact actgcactcc agtctgcatg atagagtgag actctgtctc caaaaataat 18600 aataataata ataaaataaa gaacgg.cagg ggggagacaa atatacacac agagagacag 18660 aaagaaacaa aggcaaagag aaattgaggc agagaaaatt agagagacag acagacaaag 18720 cttaggaaaa ggtctgcaga ggaatgagag aagacaggca agtgagaacc agagagaggc 18780 tgcactaacc tgatgttctt gggtccttac agaccactct ccctccagct ggggccagtg 18840 ctgagcctgg tgtatacagg tatcacttaa caagtacaga ataattccca gaagactgga 18900 gagccctaga tgtggaaaga agagattaag ggggagtaat aggtaggggt ggaaagatgg 18960 ttttttattt gttttaaatt agagacgggg tctcactctg tacccaggct ggagtgcagt 19020 ggcacgatca tagctcactg cagcctcaaa ctcctgggcc catgtgatcc tcccacttca 19080 gcccctggac tatttcaact gggactacag gcatgtgcca ccatgtctag ctacattttt 19140 tttttttttt ttttgtagag acagggtctc cttatgttgc ccaggctggt cttgaattcc 19200 tgatcctttt gaatcaggct cccgaagtgc tgggtttaga ggtatgggcc cctgtgccca 19260 cccagggctt tttaatttat ataagcaatt gattgaacac ctactctgcc cagcccctat 19320 ccctgggatt taactgtact cactcccaga gtcagaggtg gggcctgaga ggaggtgcag 19380 agtgagaacc ggctgcatgg actctatagc tgtgttgcct gggtctaaat cctggcctca 19440 gtaatgagta gctgtgcaac tttggtcaaa ttactcagcc tctcggtctg cccatctata 19500 aactggagct aataatcaaa ttgcatctgc ctcacattgt tgtagtgaga gttcaatgga 19560 attacgcgtg acgtgctggt acataattag ctgttacggt tattctcatg tttaccatta 19620 ctgagtgatg gcagacaatc acacagagat aggtgacagc ctgatgttcc ccaggcactt 19680 cagtctgtgt ccttgacctg CtgCttCttC CtaggggCCC tcatggaccc caccttCCtC 19740 ttccagtcca ttaccgccaa catcatctgc tccatcgtct ttggaaaacgattccactac 19800 caagatcaag agttcctgaa gatgctgaac ttgttctacc agactttttc actcatcagc 19860 tctgtattcg gccaggtcag gg,agacggag agggacaggg ggtgtggggg tgaggtgaac 19920 acccagaaca cacgagaaaa ggatgacctg tcttgggggc tcagaaatgc agcttatcct 19980 tggaagaaac gcagacatgt gaagaatcag ggacatggag acctggaggg aggagagacg. 20040 gtgagacagg gatagagaca ctgagagaga gaatgaggcg tgatggggag gcagaaatag 20100 agtcagagag agactgagag aaggaagatg agcaaaaaca agacaaagaa gagcagaaat 20160 caagagattc tgagagacag agttgatgag aatgagtgtg aaagagaggg agagagagag 20220 aacgaataag gctttgggct tcatgtctat tctgctcctg. gatgtcattt etgttttatt 20280 ttttttagac ggagtctcgc tgtttcattc cagctggggt gtagtgttgc catcttggct 20340 tactgcaacc tccacctccc gggttcaagt gattctcctg cctcagcctc ccaagtagct 20400 gggactgcag gcatgtgcca ccacacctgg ctaatttttt tttttttctt ttcgagacag 20460 agcctcgctc tgttgcctag gctggagtgc agtggcacaa tctcagctca ctgcaacttc 20520 cacctccctg gttcaagcaa ttcccctgcc tcagcctcct gtagctggga ttacaggcgc 20580 ctgccgctat gccaggetaa tgtttttgta tttttagtag agacggggtt tcgccatgtt 20640 ggccaggctg gtctctaact cctgacctca agtgatctgc ccgcctcaga ctcctaaagt 20700 gctggaatta caggtgtgag ccaccatgcc cagactgctt ctggttcttc tgtatccttg 20760 cttctcagtc tttggtaaag ctctccacct aaagaaaatg aaggataaat gacaataagg 20820 aacagcattt cttcattttc tcccatttct ccttctccct ctgtgttttt ttttttaact 20880 ttccccagat tgtaaaggca gtcttctgct cttttaaaac aaaatactaa aatgtctcct 20940 tatttattaa cctggaaata tgcctattac atattaaatt taagaatatc aagctgcaga 21000 acagtatgca tagctgtagt ttgttgttgt tgttgttttc agacagtatc ttgctctgtt 21060 actcaggctg gagtgcagta gtgtgatctc aactccctgc cacctccacc tcccaggttc 21120 aagcaattct catggctcag cctcccgagc agccgggact ataggcgagc gccaccacac 21180 ccagctaatt tttttgtatt tttagtagag atggtgtttc accatgtcgg ccaggctggt 21240 caacatagct acagctatta agcagggatg tatgttggat tcacatgtgg ggttgtcaca 21300 gttatggatt ttcaggaccc tactttctgg gcggtctgat ctggaaagtc tgggatgggg 21360 cccaaggtga gtacttgtaa caagcccgac cagtaattct aatgttctcc tcccaccgag 21420 aaccacagag aaaagtctgg aaggagaccc accagacagt taacaatggt tatctctaga 21480 aggagagatt aagaaggaaa tttacatctg actatatatg tttgcatttt tgcaattatt 21540 tgcaataaat taggcattcc attcttcatc aaagtaatag aaataacctc caaaatacgt 21600~
agtcctaaca tgtcagcagg.cttatcttgt gtaagaatca ttttattaat atctgacaca 21660 gcaagggaga tgaggagagg tgggaagagg gagagaaaag tatgagaaag acaaataaac 21720 aggctgaggt agacaatggg tgacacagaa aggaagtgag acagagacta agagagatag 21780 aaaggagaga ggcagggaga tggggcagag gccaagaaaa agacagaagg atgagggagg 21840 aagatgcaga aagagg.taaa tgtgagatag atcaaaggag atatagagtc agtgagtgag 21900 gggttcagag gcagagggga gtggggaagt ggggttccca tggagggatt ggggcccagg 21960 aggcgctctc tccctgtgac ctgctagctc agccctaggc aaacctcacc accccttctt 22020 tcttgcagct gtttgagctc ttctctggct tcttgaaata ctttcctggg gcacacaggc 22080 aagtttacaa aaacctgcag gaaatcaatg cttacattgg ccacagtgtg gagaagcacc 22140 gtgaaaccct ggaccccagc gcccccaagg acctcatcga cacctacctg ctccacatgg 22200 aaaaagtggg gtctgggaga ggaaaaaggg aagggagggg agggagggca agatggagag 22260 gtgagaagag ggagggaaaa ggggtaggga aggggaagat ggggaggg.aa gaagaaagac 22320 tagggagggg agaataggga aagggaggag agaacatgag gaaggaaaga aagatgaggt 22380 gaaaggaggg agaaaatagg gaggaggaac tgagacaggg agagagggga ggtgggaaga 22440 cagaatgaaa gacagaggga gagagagaga agactggctg aggaaggaat tcggggcaag 22500 ggacaaaaat acagcaacaa gagaaaaaac tcacagaggc agaaagagac ggggacaaaa 22560 agagagaaac acatcaaaga gatgtggaga gagatagaaa cagagttagg aagactaaag 22620 agaggctgag agagatgagt tagagatacg cggttggatg tgtagaggac agagaaaagc 22680 aaactgggcc agatagtgtc aaagaccttt aggccaacgg agggcagcca gggagatggg 22740 cgtatacaca gcaaggctac agcctcccct gaccctcccc ttccttccct actgtggacg 22800-caggagaaat ccaacgcaca cagtgaattc ag.ccaccaga acctcaacct caacacgctc 22860 tcgctcttct ttgctggcac tgagaccacc agcaccactc tccgctacgg cttcctgctc 22920 atgctcaaat accctcatgt tgcaggtggg ccagggacag ccagtcaagg gggtcttctg 22980 acctccttct gagctgcaga aatggggcta tgggtaccac ctggatgaga gaggggatgc 23040 tggcttccta ttctgggagc actgtaggct ctgggctaga ttccaaccaa gccaattctg 23100 ttggtggatg catggatgca tgaagaatct gtccatgcgt tctcccactg ttttcttcca 23160 tcacttaagg attttttgtt ctaaggtttt tgtttgtttg tttgtttttt gttttttggt 23220 tttttttttt ttgtcttttt tgagacagag tctcgctctg tcacccaggc tggagtgcag 23280 tggcatgctc ttggcttact gcaagcttca cttccagggt tcacgccatt CtCCtgCCtC 23340 agcttcccga gtagctggaa ctacaggcgc ctgccaccac acccggctaa ttttttgtgt 23400 ttttagtaga gatggggttt aaccatgtta gccaggatgg tctcgatctc ctgacctcat 23460 gatgtacaaa tttagggggt acatatgcag ttttgttaca tgcgtaggtt ttgtaatggt 23520 caagtttggg ctgttagggt atttatcacc caaacagtgt acattgtacc cattaagtaa 23580 tttCtCatCa ttC3CCCCCC ttCtgCtCCC tC3CtCttCt gagtCttCaC tg~tgtatcat 23640 tcctctctct gtgtccatgt gtacacattt tgtagcactc actcatgagt gagaacatgc 23700 aatatttgac tttctgtgcc atcacttaaa aatcgatcca tccacttatc atttcatcta 23760 ttcattcttt aattcattaa ttaaagaatg tataattact gattctttca tttatgattc 23820 atctaaggac atatactgtc attcatttat ttggattact tgatccacaa gttgatcctt 23880 tgaaacagtg gtatgttgat ggactatttg tcattgattc attggctcat tcattcattc 23940 attcatgcat tcatccatcc ttctacgaac taggtttcac tcttatcctt ccatg.agtca 24000 acctttcaat tcacccttaa tccatccgtg atttccttca tttatcacaa taattcactc 24060 atttattcac ctctgatcta cttatcattc aatccagcct ttcatttact cctttattta 24220 ctcatacttc actaatttaa ttattcactt gctcttccat aaatctagcc attcatgtga 24180 ttattcatta attgggttca ttgatttctt tgtctatgga tcattcatta gtgattaatt 24240 aatcaatcca tctattaatt gataagtaaa tacagacatc catttattgg tttgttcatt 24300 tattcatcaa tctttccatc catgaattga tctattgatt gattgattga tgtttttatc 24360 cagttgttca tgaattcatc tattgttcta ttacactgtt atatacctga ggaccaagaa 24420 tagtgtctca actatattat aaacacaata aatattagtt cattttctac tcatcttaga 24480 gagggtgttt tgagaggttt gtagcctgga gttcttaatc tgaaattcca tgcaaaatcg 24540 tggtgtgtgt gtgcatgtgt gtgtgtgtac ctgggcatgt gggaagagga tctgtaatat 24600 tcattagatt tcaag.cagtg agaattcttg ttgcttcctc cctcctcccc tcaccccatg 24660 ctgattactt tgaggggtat caaggatcta accctttact ataggttttt cattggtcaa 24720 tagaaagtag tgtccttgct gaaaggtctc tttttaaaaa aatttttttt tcttttgaga 24780 tggagtcttg ctctgtcacc cagtctagag tgcagtggca tgatctcggc tcactgcaac 24840 ctccacctcc tgggttcaag tgattctcct gcctcagcct cctgagtagc taggaataca 24900 ggtgtgcacc agcacaccca gctaattttt tgtattttta ggagagacgg gattttgcca 24960 tgttggccag gctcatcttg aactcctgac ctcaaggaat ccacccacct caacctccaa 25020 aattgctggg attacaggca tgagccacca tgcctggcct gaaatgcctc tttaaaatga 25080 gattcattgg tcttcttttc tgtacagaga gagtctacag ggagattgaa caggtgattg 25140 gcccacatcg ccctccagag cttcatgacc gagccaaaat gccatacaca gaggcagtca 25200 tctatgagat tcagagattt tccgaccttc tccccatggg tgtgccccac attgtcaccc 25260 aacacaccag cttccgaggg tacatcatcc ccaaggtaag accggctgga accccatagc 25320 cctcctgttt gggcatcctg gattctctta atccccgaac tcaacctttt gttagctcct 25380 taattgagtc ccgttgtttt tgttttttgt atttcttttt tgtggagtgt gtggagggtt 25440 ggagggaatg gcaatatctt ttgatcttgt gatcctccct caggacacag aagtatttct 25500 catcctgagc actgctctcc atgacccaca ctactttgaa aaaccagacg ccttcaatcc 25560 tgaccacttt ctggatgcca atggggcact gaaaaagact gaagctttta tccccttctc 25620 cttaggtaag ctggacccac aatttctttc ccagacacca gagggcaggt actatcecca 25680 acttgagaaa aacaacgaga gatactgatt atttgagcac ttaatatatt ctgattgctt 25?40 cacctgcctt atcccattcc atcttcacta caaccctata aggaggcttg agaaagaaga 25800 ttacattccc aaaggcacat cttggcaagc aggaccttgg gcaagtattt taacatctct 25860 aaacctcagt gagttcattt tcttaaaaag aaaaaatctg ttgggcacca ctgtaagccc 25920 agtgctgtac tgggggctga agataatgca tcaaacaagt cacacagaga cagggttcct 25980 gccccaggaa atttaaagtc cagcagggaa gatgggcatt catcaaataa taataaaata 26040 atcatctcat gaaatgaatg aatgcctgca acatgcttag aactgcctgg cacaaaggac 26100 atgctcacaa gggcaattat tattataatt agacataatt gtgataagtg ctctaaaggg 26160 agctttggga gcacaaaata ggaaatagta g.ctaatcttg tggtgggtcc gtaaggaaaa 26220 gcttaccaga ggaactggct tccaagctaa catgttcatg ggtgagcatc aatcaaccat 26280 tgcaaagtgt gttccaggca aaggaaacag caaggacaaa ggccaaaggt agaaacgtga 26340 tatggcacca ttgagaacct ataggaagtc cagtgagcct tgggtgtaga ttgcagaagg 26400 aaataagaca aagggctcat ctgggcagga ccttgaaggt tgcaggagga gtttggattt 26460 atggtctagc actgggaagg tggaaaaggt cttgacgtgc tctgacttgt cccagttctc 26520 atcctctact ctttggctgg ttaaaagaaa aactttagac~aaattaaact tagcagagtc 26580 tatctgaaca aagaaacaat tcatgaattg ggcagcacaa ggaaccagta aaggttcaga 26640 gagctccatc cagcaatgtg gtcaggcact atttatccac agggaaagga actgaggtac 26700 tgaaacagcc tgattggtta cagctctgtg tttgccttat ctgagcatgt ctgggcagct 26760 tgtagcctgt gactggctga agcttggctg ccctgattgt ccaaggttac ttgttacaag 26820 aatatactct caagtttgtt tacatgttga gttacattac aatttgttat gtagggaggc 26880 tgctttaggc caaatttaat tgaatttaac atgcaccatc catgaaaggc cccagaaaca 26940 aacccccagt cttgagtttc atcagtgact tcagtgatcc agggtttggc ccagcccccg 27000 gttttgcgca gtacagtgac ctccttccac attttatctt caatgaaaat tgggaacatg 27060 tgtggtgatg gcttgaatgt caatgtcttc tctcaatttt gctatctgaa gtttcttttt 27120 ttttttttgt cttcagacgg agtcttcctg tgttgcccag gctggagtgc agtgtcacga 27180 tcacagctca ctgcaacctc tgcctcccag gttcaagtga ttctcctgcc tcagcctccc 27240 gagtacctga aactacgggc gcatgccacc atgcctggct attttttttt atttttagta 27300 gagactgggt ttcaccgtgt tagccaggat ggtgctgatc ttctgacctt gtgatatg.cc 27360 cacctcagtt tctgaaagtc ctgggattac aggcataagc cactgcgact ggcctgctaa 27420 tttgttttaa gagaggcaga gatgtaggga aataaggaca gagacagata agtaacaaag 27480 ataatgagac tgag.aaacag agttagggac aaagagggaa cacatagaga tagagatggg 27540 gagccagtgg cagaaagaca gagggcaaac ctcagacagt atacagagag gaagagagag 27600 agacacagag agagagagag tcacgtaagg agaaggagga ggaggaggaa taagaggaga 27660 gagagtagga aaaggaagga ggaggggaga aaggagggaa aggagagaaa cagaataaga 27720 gataggaaaa agagagagtg acataaaaag agaggaagaa agaatgaaca agacaaataa 27780 ggtatcttta gagggagatg aaaagaaaga atgagagaga aagagattgg ggagaaatca 27840 gattcaaata gatggagata ggaagttaag caagatggaa gaaagccaaa aaaaggagag 27900 gaaaagaaga aaag.ctgtgt ctgacaggta tagacagaga aaaagacagg agtagggctg 27960 ccaggggcag aaagaaaggt ataacccaag ccaaaagggt actgcagcca aagaaattcg 28020 aaaggtgtcc aaacagacgg ccccagagat ggggagggcg tgatgagagg gagataataa 28080 gcctcaggct gttgtgaaaa atgttaggtc acacaaagag ttacagaaga acaggcccag 28140 agacctgcct gtttctaagc tcatgtcacc caccttctgg gtatgccaaa gggatgtgga 28200 cacttttcca aacacctcca catagacaca cttgtccaac aacttgacag gcatagggaa 282-60 tgcctgaaaa ctcacacttg acatggcctt tccaaggttt gcagattatg aaacactgaa 28320 gtgaagggaa aggctccctt tgtctctgcc tgagcttttt ccagcaccct ttgttcttat 28380 ttttctccca atctgtggtt tagaatctac tgggggttcc ttgcacctct gagaatcagt 28440 ggaagccata gaccctccct gtctcatacg catcagtccc attcacaatt tatatacaat 28500 tgggcttttc cccccttgag ctcttgaagc ctgtggatct caggctgaga ctagaagaca 28560 gcctagagac acagacacag acacatggcc agaaacacat accccacaca taccccagaa 28620 acaaacacaa ggcagtaacc aaaccttaag cctccttagc acaagccaag tgctattcta 28680 agaattttta caattgtctc ctttaacctc acaagagccc tttgagggag gtgccattct 28740 ccccatatga gaagtgaaga acactaagaa tgttgtcata aatgtgctca cactcacaca 28800 tgtacaggta cacacacaca cacacacaca cagaaccaga aatgggtcat tatactctgt 28860 gggtttcacc aaccctttgg aactgtaaca gaagctagaa actctcttat ataaaaatga 28920 acatggcgcc gggtgcagtg gctcatgcct gtaatcccac tttgggaggc caaggcaggt 28980 ggattatctg aggtcaggag ctcgaggcta gcctggccaa tatgatgata ccccatgtct 29040 actaaaaata gaagaaatta gccaggcgtg gtggcaggtg cctgtaatcc cagctacttg 29100 gtaggctgag gcacaagaat catttgaacc acctgggagg tggaggttgc agtgagccaa 29160 gatggtgcca ctgtgctcca gtctgggtga caaag.agaga ctccatctca aaaaaaaaaa 29220 aaaaaaaaaa aaaaaaaaaa aaaagagaga gagagaaatg aacgtggcac atccactcaa 29280 agatttgcat ctggtttcag agcagcttcc taaaagtcca ccctgaattg taggttaaag 293'40 gccagtctta tgcaaatctg ttgcagtgga catttgtgtc tgggcttagg gacatggcag 29400 agcgaagtgt atgcacctgc cctgtgccca cactggtgac cttctgtgtc cacagggaag 29460 cggatttgtc ttggtgaagg catcgcccgt gcggaattgt tcctcttctt caccaccatc '29520 ctccagaact tctccatggc cagccccgtg gccccagaag acatcgatct gacaccccag 29580 gagtgtggtg tgggcaaaat acccccaaca taccagatcc gcttcctgcc ccgctgaagg 29640 ggctgaggga agggggtcaa aggattccag ggtcattcag tgtccccgcc tctgtagaca 29700 atggctctga ctccccgcaa cttcctgcct ctgagagacc tgctacaagc cagcttcctt 29760 cccctccatg gcaccagttg tctgaggtca cattgcaagt gagtgcagga gtgagattat 29820 cgaaaattat aatatacaaa atcatatata tatatatgtt cttgtttttt gagacagagt 29880 ctcacactgt tgcccaggct ggagtgcagt ggcgtgatct cggctcactg caacctccac 29940 ccccggggat caagcaactc tcctgcctca gcctccctag tagctgggat tacaggcatg 30000 cactaccacg cttggctaat ttttgtattt ttagtagaga tggggtttca ctgtgtaggc 30060 caggctggtc tcgaactcct gaactcaagt gattcaccca ccttagcctc ccaaagtgct 30120 gggattacag gcgtgagtca ccgtgcccag ccatgtatat atataatttt aaaaattaag 30180 ctgaaattca cataacataa aattagctgt tttaaagtgt aaaatttagt ggcgtgtggt 30240 tcattcacaa agctgtacaa ccaccaccat ctagttccaa acattttctt tttttctgag 30300 atggagtctc actctgtcac ccaggttcga gttcagtggt gccatctctg tccactgcaa 30360 cctccacatc ctgggttcaa gtgattctcc tgcctcagcc tctggaggag ctggtatcac 30420 aggcgtcccc caccacgcct ggctaaattt tgtattttta ggtggtcttg aactcctgat 30480 gtcaggtgat tctcctagct ccaaatgttt tcattatctc tcccccaaca aaacccatac 30540 ctatcaagct gtcactcccc ataccccatt ctctttttca tctcggcccc tgtcaatctg 30600 gtttttgtca ctatggactt accaattctg aatatttccc ataaacagaa tcatacaata 30660 tttgattttt tttttttttt tgaaactaag ccttgctctg tctcccaggc tggagtgcta 30720 tggtgcaatt tttgttcact gcaacctctg ccttccaaga tcaagagatt ctccagtctc 30780 agctcccaag tagctgggat tacaggcatg tactaccatg cctggctaat tttcttgtag 30840 ttttagtagg gacatgttgg ccaggctggt ggtgagctcc tggcctcagg. tgatccaccc 30900 acctcagtgt tccaaagtgc tgatattaca ggcataatat gtgatctttt gtgtctggtt 30960 gctttcatgt tgaatgctat ttttgaggtt catgcctgtt gtagaccaca gtcacacact 31020 gctgtagtct tccccagtcc tcattcccag ctgcctcttc ctactgcttc cgtctatcaa 31080 aaagccccct tggcccaggt tccctgagct gtgggattct gcactggtgc tttggattcc 31140 ctgatatgtt ccttcaaatc tgctgagaat taaataaaca tctctaaagc ctgacctccc 31200 cacgtcaaga ggtg.atctgt gccattttgt gtgtgattct tttattgtcg ggtctctagg 31260 gatttttctg gaaggaatgt tggtgagaat gcctctctca cctcaatgcc aactctgtga 31320 agggccaaac cattgtcttg ctcatccctg tactctcaac acagcgtgtg gcatatgaca 31380 ggtgttcaaa atatttggtg aggaatgaat gaatgagtgg ctaaatcagc caccccctac 31440 ccccacagcc cacccaaaat ggagctaggc ctcctccatc agactatatc tcctccatat 31500 cccactttct tgtgaggtcc agaataaatt ctcatcccca aagggcccag gatgccccca 31560 ttctgcctac cagttactct cgataccccg tgctgcaatg ccaccttttg ataaagctta 31620 gcgctattct tgcagtggaa ccactctaaa tccagcctcc aggctggacg cggtggctca 31680 tgcctataat cccagcactt tgggaggccg acgcgggtgg atcacttcaa gtcaggggtt 31740 cgagaccagc ctgaccaaca tacaaaaacc gtgtttcaac taaaaaagat acaacaatta 31800 cccgggcatg gtggtgtgca tctgtagtcc catctactcg ggaggctgag acagggtg.at 31860 cgcttgaatg caggagggag aggttgcagt gagccaagtt cccgccactg cactccagcc 31920 tgggtgacaa agtgagactc cgtttcataa ataaataaat aaataaatcc agcctccaaa 31980 cattccacca gcaggaccac cacccttccc tgagggaggt gctgcaagtt taatatcatt 32040 atcacaagga tacatcagcc agcctgtggc cagtgggaag ctggccctgg gactccaccc 32100 tgagatgcta accttttggc gtctgcagca tctgccatct ttgggctcct tgtagcctct 32160 gaagagttaa cagagactct gccagtacaa atgcatggca cacattccct caccaaccac 32220 agaaataaca aacagagcaa catgtctttt aagagcgaca gcagaatgga aggcgggaag 32280 aatgaggacc acggtgccct cagccttctc actcctcatt gtccccagtg gcctcttgct 32340 actctggttc tggggccatc ccacctccca gggctgcctc tCtCCtaggC CCtggCCtCt 32400 gcccttcctg ggggagcatt ttgcaaataa accacagggc tttctgaagt ctgtccaggt 32460 ggtgagatgg gagagtgtgg aagggatgga aaaggaggag gtgggtagac gggaaacagc 32520 tcacaggcag caaggcagtt ggggtcgtac tttgggtggg attccacaga caagcttggg 32580 aattcctttc tggcttatat attaactaca gaagcttctg gaattttata aggtgaaatg 32640 gagaggagac agactggagg gtgaaattct gatagacttg aggctttgag atgtggtcct 327-00 ggggtggagc aagacaagaa aagtactgga gattggggtt tgaggagtct atgcaattat 32760 ttttattttt aaaaatcttt gtggctacat agcaggtgta tatatttatg tggtaagtga 32820 gatatttcga tacagacata caatgtataa tcacaggcat acaatgtaga caggcataaa 32880 gtgtatagtc acatataata ataacatcat ggtaaatggg gtaaccatca cctcaagcac 32940 tcatcatttc tttgtgttac attagagtta tattccctct gttatgctaa aatgtaccac 33000 aggctgggca cagtggttca tgcctataat cccagtattt tgggaggctg aggcagacag 33060 attgcctgag ctgaggagtt cgaaaccacc ctgggcaaca tggtgaaacc ctgtctctac 33120 aaaaatacaa aaaaaaatta gccaggcatg gtggcatgca tctgtaatcc cagctactct 33180 ggaggctgaa gcgggagaat tgcttgagcc tggtaggtgg agtttgcagt aagccaagat 33240 tgtgccactg cagtcccgct tgggtgacag agtgagactc catctcaaaa aaataaacaa 33300 gcaaacaaac aaaagtacaa tgaattattg ctgacggcca tcaccttatt gtgctatcaa 33360 aaactagatc ttattcattg tatcgaactc tatttttgta cccattagcc atccccagtc 33420 cctcacctcc acccttccca gtctctggta accatcattc tactctatct ccatgagtta 33480 aattgtcata atttttagct cccacgaatg agtgagaatg tgcaaagttt gcctttccgt 33540 gcctggctta tttcacctaa cataatgtcc tgcagttcca tccatgttgc tgcaaatagc 33600 aggatatcat tcttttttaa tggttgaata gtactccctt ctgtatgggc accatatgaa 33660 cacttaggtt gattccataa cttggctctt gcaaacagag ctgcaaaata cactggagtg 33720 cagatatctc ttcagtatac ggatttcctt ttttttgata tagatttagc ggtgggattg 33780 ctggattgtg tggtagctct atttttagtg gttttctttc tttttgtttg tttattttat 33840 ttctttattc attttttttg agatggagtc ttactctgtc acccaggctg gagtgcagtg 33900 gtatgatctc agctcactgc agcctccacc tcctgggttc aagcgattct cctgcctcag 33960 catcctgagt agctgggact acaggcacct gccgacacgc ttggatgatt tttgtaattt 34020 tagtagagat ggagtttcac cacgttggcc aggctggtct tgaactcctg acctcagatg 34080 actcgcccac ctcagcctcc caaagtgctg ggattacagg catgagccac cgcacctggc 34140 ctattctact tttttttttc tttttcttag ggaaactcca tgttgttcct caaagtggct 34200 ctattaattt acattaccac caacagtgta caagggttcc atggggatta cttcagattg 34260 agtggtcaga gagggctcct ttgagaagac ttctgagaat tggccatggg aaggtgtggg 34320 gagaagcctt ctcagctgag ggaacagcaa ggtcaaagac ccagaggtaa gaaagcaagc 34380 ttggaacctt ccaggagcaa caaggcattg gctctgataa tgccttctcc tgctaaaatg 34440 acagctctag gaaggcaggg ttcttatttc ctgtctatct atctatctat ctatctatct 34500 atctatctat ctatctatca tctatctatc tatcatatat ctatctatcc gtctgtcttt 34560 atttatttat agaggcagag tcttgctcta ttgtccattg tattagtcca ttttcatgct 34620 gctgataaag acataccgta gactgggtaa tttataaaga aaaaaagttt aataaactca 34680 cagttccaca tggctgcgga ggcctcacaa tcatgctgaa aggcaaaagt tgcgtcttac 34740 atggtggctg gcaagagaga gaatgagaac caagagaacg gggtttcccc ttataaaacc 34800 aacagatctc atgagactta ttcactacta tgagaacagt atgggggaaa ccacccctat 34860 gattcaatga tcacccaccg ggtccctccc acaacacgtg gggattatgg gagctgcaat 34920 tctagatgag atttgggtgg ggacacagcc aaaccatatc accaaggctg gagtgcagtg 34980 gtatgatcat ggctcattgc agcctccaac ttctgggttc aagcaatcct cccacctcag 35040 cctcctgatt agtggtgact acaggtgtgt gacaccacct ctggctggct tttaaatttt 35100 ttggagagat ggagtcttgc tattttgccc aggctggtct caaactcttg gtctcaaatg 35160 atettcccat cttgacttcc caaagcactg agattacagg tgtgagccat tgcacctggc 35220 tggttctcat ttatttaagt attactgctg tcatggtttg ctttggtccc tgtgggttcc 35280 cccacccttt gaaacaag_tg cctgacacat ggtagggttc acaataaata tttgtagaat 35340 aaataagagc tgagcettca agggg~tccaa aatacttgca cggagttttt ctaggtgggc 35400 acgatgccac ccagctgcag gactatttca gggtgttcat tggctcctct ggttgatctt 35460 ttgatgcctg tataagaact cccacttctc caaatctggg ggccacatct ttagagttct 35520 cagctttctg tctctcagga atgaaggaga taatatcact gattggagcc agattgtctg 35580 gatgttgaat cccagctcct tgctggtgcc tttgagaagt gactttagcc ctccatgtgc 35640 cttatttctg cctctgtaaa cggagatcat catagcacct ctcatgtggg gctgtttgac 357.00 gattcggtga ggtatgtgca aagagctttt tgcacttgca aaaagtgtct gggatatcat 35760 tgtgtgctgt aagaatgagc tgtaatgatt atcactgaga cttgttgttg ggattaaaag 35820 agatcaggtc tgtcagggcc tggtacacaa gaggtactag aaaaataatg ggaggtttag 35880 gtgctccttc ctccagatac actcgtaaat atgactgctg tgtcgtcatc tactttttag 35940 ggatatgaag aatctgctgt gtgaccttag atgaataaat ctctgattca ctctgggcct 36000 tggttttccc tgcctctgaa aggggtatga tgtttccttc ccttttgcct gggcgcaggt 36060 actgggtgct ttgtagttaa gggagtatca gctggtggct caagggtaga ttctaacagg 36120 aacttccaag ctggagaaat actaggctac acctcagaga ggggaggtgg gtaagggagg 36180 cccctgctgg tgtgagcagg aggtgggtga gggaggcccc tgctggtgtg agctcgatcc 36240 tttatccctg agcttcagac tctcttaagc cttcccacat gagaaactga catccagatg 36300 gaggtgggga ccctgcaatg taaagccagg tcacctgtcc acagaggtgt gtctgataac 36360 cagggcatga taggttagag. agtgtggtgg ctaaaagcac cagcccttgg atcacctgag 36420 gtcaggagtt caagacctgc ctggccaaca tggtgaaact ccatctctac taaacataca 36480 aaaattagct tggtgtggtg gtgggcacct gtaatcccag ctacttggga ggccgaagca 36540 ggagaatcac ttgaacccag gaggcagagg ttgcagtgag ctgagattgc gccatttcac 36600 tccagcctgg gtgacaaaag cgaaactctg tctcaaaaaa agcaccagcc ttagactcag 36660 agagaccagg atttgagctt gggggacctc agcttcctct ctgtgtgacc tcaggtaagc 36720 cacttatact ctttgggcct cagtttcctc atttgtaaaa ttgaggccgt gattgaacct 36780 ccttcaagag ctgttgtgag tgctataggg gatactatct taaagcactt aatgcagtgc 36840 ctggcacatg gtagttctgt agaagtgctt gctattttaa ttcacgtgca caggtcctgc 36900 ctgtgggtga gggctcagta gatggaacag cgatggttga tatagttggg attgtgagga 36960 ttattatgac aggggctaat ttgaatgggc ctgtgttgtc tgccctcccc actgccagcc 37020 tgatttccct ctgcctggct ttgcagctcc tcccattggc tgctggggtg taccccttgc 37080 atgcggatga gaaacgagtc aatgaagtcc cgtggggaat tgggatccag cgtgcactgg 37140 ttgtgctcca ctttcttggc aatgaagtcc tccagccctt gcagctcctt aaaggcctgt 37200 tgctgtggtc ctggcaggtg tttcatcacc gaagagaaca tctcatagag ctggggttgc 37260 agagagagga tgggaaggga aggaccgctg tcaggaggca ggaactgatg ggaatgggac 37320 ctgtgtcaga gcaagaaagg cactgggtta aaggcaccta cctgcccagg tgtttaggta 37380 tttcagtggg gctggatggg aacatatatg taagtcttca tgagggttaa gtgagaaggc 37440 tactgtccag aaatttaggt aacatggact gaggcacaca tccaggtttc aatgaaggtg 37500 gattggagcg cacagctagg tgttcagaca ttcaaggcag gatgtgttgg aaaacacctg 37560 ttcaactgtt tagctactca gacggggctg gtatgagggg agtgtcacct gtctaggtat 37620 gtaggcattc aggtgggcct ggttgaagac aactaccgag atacttattg ggtctggatt 37680 gggggctttt gttcagatgt tcaggtatta aagtggggct ggtgaagatg ttgaagagga 37740 ggggatacct gttgaggtgt ttacctatcc aagtgggatg cactgggggc acctctgtaa 37800 ggtgtgtgat tggaaagggt tggggaacac ttatttgggt gtctggggaa taatctgtct 37860 agatatttat gtgtttaggg tattggttag ggcacctgtc catgttttca ggtatttaag 37920 tgggtgaagt agagggcgct tggccagata ttccagtggg ctaatctggg acatttgtgt 37980 agatgtgaaa tgattatgat gggctggatt ttgcagcacc aggtgttcag gtatgcagga 38040 ggcgggttga ggcagtcact tgtccaggtg ttaaaatatt cagaagaact gggtagggag 38100 catctgttag aaattatggt aagttgggga tgggaacatg tgcccaggtg. agagagctga 38160 gctgagggta tgcatcctgc cttcaggctc tgttttgggg atcatctgtt gagctatcca 38220 ggtgtccttg gagactgggt attgggcacc catcctgggt tctggtgcaa ctttccggtt 38280 gtccaatatt gggggctgat tttgagggga cactgtctgg aggggggtgg gagtttgggg 38340 cacctgtccc catgtagggg agcagttggc aggttgtggt aggggcgtga cagccaggct 38400 gcagccggtt acctgccccg tggagggtgc cgtgaactgg aagctttcca gcatcatgca 38460 cagcagtgac aggaactttt gtcctcatag tcaaagcggt ccccaaagac aatggagctg. 38520 atgacattgg aggtggtacg gcttaggaag aaggtgggat agatgctgga gcttgcgggt 38580 gtggcggtgt attgggggag aagggggttg ggaagagagt caactcagag ctctgatgct 38640 agtctgagaa ttccaggagg cactgccctg. atgagccaca ttcccagcgc cagactacag 38700 ggctgggagc gcgggcgcct ttccccacca aggcccggtc cccagacaga acgcgcgcgg 38760 gttcctcgcc ctgggcgttt tccttctccc gcctccacac tcggggtgtt ctgctcaccg 38820 cgcgtgccct agacggcctt gatgaggaag cccgccttct cctggatacc tccttgatgc 38880 cgcgcttgcc caccccgaag tcecgcaggg tggcgatgga gaagcgcctg agcggcttgg 38940 tgCgCtCCCa ggtgcggcag gtcactcctg ggagaggaac gcaggggtgg ggagaggtca 39000 ggccgtggga acaggcacag aagcctcgcg caggagaact gcaaactcag~ tcagag~aac 39060 acagagagaa acagagaccg aacaggaaca agaggatcag aaaagagaca ggccagacag 39120 acagatgcac agcagaacag agggagacgg gacactccat ggagcatagg, gagggaagga 39280 gggagagaaa aagacagaga tgggagaaga tgatggaagg~ gggggaagtg aggtatgggg 39240 agatctgcga ggagaaaata aaaatggtga aagagtcttt tttgtttgta tgtttgttta 39300 tttgtttttg agacggattc tcgctctgtc gcccaggctg aagtgcattg tcgccatctt 39360 ggccgctcgc tgcaacctcc accttccagg ttcaagcgta aaaatggtga aagactccta 39420 agctgagctg gtgaagacag aaaactacgg aaagagaaac agagacggtc gggaaggagg 39480 agagactgtt atcaggagag gaggctggag atgtggaagt aggaataccg cgaaatccta 39540 agacagagga agaagaagga gaggtcagtg gacagaggag aaactcagag aagcacagaa 39600 gctgagaagg acaaaaagac aaatgaagac agagaaccag ggttggaaaa cagaaaccca 39660 ggattgtcct cagagatggg gaaagaggag actccagtga gagaggcagg gagaaatctg 39720 gacagggaca gggacaggga tgctggagac aggcaagagg gagaggccgc aatgaaggga 39780 gctggggagc gaaggccaga cagggggctc tgccgcagcc ttcagtgggc aggagagtca 39840 gggagaaggc tggggacacg gaggccatcg agtcggcctg gccaccttcc ccctcttggg 39900 caccccctca ccatagcctt tgaagagcca gtcgaaggtg gcctgctcgc ctcgcccgct 39960 gaactcctca gcctggtcca ccagagcctc cttgacggca tcatgtccgc acagcaccac 40020 gacccgccgg ggccccaggt gaatggtgaa cacggggcca tagcgctcac tgatctgatg 40080 gaggcaggtg ggagtggtta gagggagcag cccccactct gaatgggcgc agcaccgaga 40140 tatcatgtac tgggatgttt tgcccccagg ttctcacagt cagggagctg gacatcccaa 40200 gatcctgtct ttccagccta ggaccctgat gctgaaactc caaaactccc tttcctaaga 40260 ctctggtcca cactggtcaa tcccctgcca caaagcctca gccaactggg cagcccccac 40320 cccgtgccac ccatctccct gccttgggac accttcatga tggagtcaca tatgtgctct 40380 gtgttcagct gcaggtagtt tccaataaag agcaatgggg tgggtcccaa aggcagcttc 40440 cccctgctct tcctctgctg ccagactgac atcaagacca tcacagtcag gctggccagc 40500 aaggccacca gaagcagccc tgaggccagc atggtggcag tggaatgata gatggtgacg 40560 gctggggtgg tttgccttta tactgcctga aaaaaagggg tggactttgg ctggttataa 40620 aatcacctca tttcacctcc caactacatc ctctccgtga accccaccta gctttggaca 40680 caaccagcaa aggaggagga gggggacccc agggaagctg aacagagagg gtacctcccg 40740 actaaatctg tggtacctca ggaggggtgc eccagggctg tggatttagg agcgggcaac 40800 agataagctg tagaacaaag gagttgggaa tatttgcata ggggagcact tgggctttgg 40860 atttgggatc tgggagtgag aatgcacccc aaaattgtgg atttgggggt tccgaggaga 40920 agaatgcaaa ggtctgggtt ttattagggt gagatggaag tgtggctgtg catctggggg 40980 tcttctgttg tggaggatgc agggttaagg gtctcaggag gggggattcc aggcatatgg 41040 acttgagagt cttggggcta ggaagaccca gggctgtggg tgtggagttt ctgggaggag 41100 gagtgtgtag caagcccagt aggtcgtccc agaaagccca gcagcccagc actgggccca 41160 ggggctgact tgattttgct tttgcccaga gctgtctctt gatgcccgga atcctcactg 41220 gaacctcaga gtgaggccag gatgggtgcc atggaaactc cactgaccat ctctggttgg 4128U
caaaggggtg gaagaccaga gccatatttc aagggaaatt tccacacacc tggagaagag 41340 taccttgtgt tatcagttgg cagagggatt ggccaggtct ggagaaatag taacaacaac 41400 ccccaaatgg tggaaaaagc tcaagtgtcc caagggttgt aatttacaaa gacctccact 41460 ggtttccaac ccagatcctt agggaaaccc actagattga ttgtatcatc cctattaata 41520 ttatgattgt tgtttatctt tctcatttta tataagggtc tacagaggac agaaaacaga 41580 cagtaggcag gggacatgca tgagtggccc tgaactccca tctctcctgc tctgagtact 41640 tagttcccct ttctggattt ggttttccca ccagtagaaa gggagtattt aggaaaagat 41700 catatttgag ggtgagttaa gtctccttta agggcgattc ctgctttgtc aggggctact 41760 actattagca ggcagaggga acaatcgcag aagagattgg ggtaaactcc agtgttaggc 41820 acagaggtag atcctggggt gggtgtgagt ggagaaggga tgagtagccc tcctgtcctt 41880 ccctcaggga aattcttcct tagtaaacgg ggcagttacc ctttggtgag catggtcgta 41340 tatgggaaga ttaagtctag gtgtgtatga tctggtcgaa tctttgtaac agtgaggttt 42000 gttcatcatg atcatcctca ttttgaagat gagcagatct agactccata aatcctcttg 42060 agtgaggtca cacagggaat aagtgagggalgctgggatgt gaactttctt ctcatccccc 42120 gtcattgact ggcctagatc accctgggag tcaagtgcag gccaggctta gcaggaggtg 42180 agtgctgaga ctggcatcag cagtggacac actgatgaca ttggcgggtt ctgatatcag 42240 catcaggtgt gttcagggag ggaaaggcat cgtgttggct ttgggactgc ccccaggtgt 42300 atgcgaggat gggaaacaag ttggtactga acttgatacc aggagaatat tgaagtcatg 42360 tttggtgcca agagggctct gtgtgttgtt tcaggtttca ttgaggg-cat aagtgtgtcc 42420 tgcttcagga gggcgtaaga atggcaccat atagggtctg aggttggtcc cagtgggctg 42480 tgacatgagc atgaggcagg tgctgatatt ggtcctatgt gggtctgaga tgagcctcag- 42540 ccagatc 42547 <210>

<211>

<212>
DNA

<213>
Mouse <400>

gaagcattgaggaggatcacacacacagttgtagggagaacacagagaagtaaattgctg.60 acaaacaagcagggatggacctggtttcagctctetcact.ggaaacctgggtgctcctag120 caatcagcttggtgctcctctaccgatatgggactcgtaaacatgaactttttaagaaac180 agggaattcctgggcccaaacctctgccatttttaggcactgtgctgaattattacaagg240 gtttatggaaattcgacatggagtgctataaaaagtatggaaaaacatgggggttgtttg300 atggtcaaacgCCtCtCCttgCtgtCaCagacccagagacgattaagaatgtgctagtga360 aggaatgtttttctgtcttcacaaaccggcgggattttggcccagtggggataatgagta420 aagctatctcaatatctaaggatgatgaatggaagagatatagagctttgctgtccccca480 cattcaccagtggaaaactcaaggagatgttccctgtcattgaacagtatggagacattt540 tggtaaagtacttgaggcagaaggcaaagaaaggcaagcctgttactatgaaagatgtgt600 taggtgcttacagcatggatgtgatcaccagcacatcatttggagtgaacgtggattccc660 tcaacaacccagaggatccttttgtggagaaagccaaaaagcttttaagatttgattttt720 ttgatcctttgctcttctcagtagtactttttccattcctgacaccagtatatgagatgt780 taaatatctgcatgttcccaaaggattcaatagaatttttcaaaaaatttgtggacagaa840 tgaaggaaagccgcctggattctaagcagaagcaccgagtggattttcttcagctgatga900 tgaattctcataataattccaaagacaaagtctctcataaagccctttctgacatggaga960 tcacagcccagtcaattatctttatttttgctgggtatgaaaccaccagtagcacacttt1020 ccttcaccctgcattccttggccactcaccctgatatccagaaaaaactgcaggatgaga1080 tcgatgaggctctgcccaacaaggcacctcccacgtatgatactgtgatggagatggaat1140 acctggatatggtgcttaatgaaaccctcagattatatcccattgctaatagacttgaga1200 gagtctgtaagaaagatgttgaactcaatggtgtgtatatccccaaagggtcaacagtga1260 tgattccatcttatgctcttcaccatgacccacagcactggtcagagcctgaagaattcc1320 aacctgaaaggttcagcaaggagaacaagggcagcattgatccttatgtatatctgccct1380 ttgggaatggacccaggaactgccttggcatgaggtttgctctcatgaatatgaaacttg.1440 ctctcactaaaattatgcagaacttctccttccagccttg-taaggaaacacagatacctc1500 tgaaattaagcagacaaggacttcttcaaccagaaaaacccattgttctaaaggttgtgc1560 cacgggatgcagtcataactggagcatgagtctccctcaaggagttcttctgagttcttc1620 agaaaggcagtgtctaagaacatcggacattttagtttcatcatgagtaaaattgagatg1680 aataaggggc 1690 <210> 14 <211> 2768 <212> DNA
<213> Human <400> 14 cactgctgtg cagggcagga aagctccatg cacatagccc agcaaagagc aacacagagc 60 tgaaaggaag actcagagga gagagataag taaggaaagt agtgatggct ctcatcccag 120 acttggccat ggaaacctgg cttctcctgg ctgtcagcct ggtgctcctc tatctatatg l80 gaacccattc acatggactt tttaagaagc ttggaattcc agggcccaca cctctgcctt 240 ttttgggaaatattttgtcctaccataagggcttttgtatgtttgacatggaatgtcata300 aaaagtatggaaaagtgtggggcttttatgatggtcaacagcctgtgctggctatcacag360 atcctgacatgatcaaaacagtgctagtgaaagaatgttattctgtcttcacaaaccgga420 ggccttttggtccagtgggatttatgaaaagtgccatctctatagctgaggatgaagaat480 ggaagagattacgatcattgctgtctccaaccttcaccagtggaaaactcaaggagatgg540 tccctatcattgcccagtatggagatgtgttggtgagaaatctgaggcgggaagcagaga600 caggcaagcctgtcaccttgaaagacgtctttggggcctacagcatggatgtgatcacta660 gcacatcatttggagtgaacatcgactctctcaacaatccacaagacccctttgtggaaa720 acaccaagaagcttttaagatttgattttttggatccattctttctctcaataacagtct780 ttccattcctcatcccaattcttgaagtattaaatatctgtgtgtttccaagagaagtta840 caaattttttaagaaaatctgtaaaaaggatgaaagaaagtcgcctcgaagatacacaaa900 agcaccgagtggatttccttcagctgatgattgactctcagaattcaaaagaaactgagt960 cccacaaagctctgtccgatctggagctcgtggcccaatcaattatctttatttttgctg1020 gctatgaaaccacgagcagtgttctctccttcattatgtatgaactggccactcaccctg1080 atgtccagcagaaactgcaggaggaaattgatgcag.ttttacccaataaggcaccaccca1140 cctatgatactgtgctacagatggagtatcttgacatggtggtgaatgaaacgctcagat1200 tattcccaattgctatgagacttgagagggtctgcaaaaaagatgttgagatcaatggga1260.

tgttcattcccaaaggggtggtggtgatgattccaagctatgctcttcaccgtgacccaa1320 agtactggacagagcctgagaagttcctccctgaaagattcagcaagaagaacaaggaca1380 ~acatagatccttacatatacacaccctttggaagtggacccagaaactgcattggcatga1440 ggtttgctctcatgaacatgaaacttgctctaatcagagtccttcagaacttctccttca1500 aaccttgtaaagaaacacagatccccctgaaattaagcttaggaggacttcttcaaccag1560 aaaaacccgttgttctaaaggttgagtcaagggatggcaccgtaagtggagcctgaattt1620 tcctaaggacttctgctttgctcttcaagaaatctgtgcctgagaacaccagagacctca1680 aattactttgtgaatagaactctgaaatgaagatgggcttcatccaatggactgcataaa1740 taaccggggattctgtacatgcattgagctctctcattgtctgtgtagagtgttatactt1800 gggaatataaaggaggtgaccaaatcagtgtgaggaggtagatttggctcctctgcttct1860 cacgggactatttccaccacccccagttagcaccattaactcctcctgagctctgataag1920 agaatcaacatttctcaataatttcctccacaaattattaatgaaaataagaattatttt1980 gatggctctaacaatgacatttatatcacatgttttctctggagtattctataagtttta2040 tgttaaatca ataaagacca ctttacaaaa gtattatcag atgctttcct gcacattaag 2100 gagaaatctatagaactgaatgagaaccaacaagtaaatatttttggtcattgtaatcac2160 tgttggcgtggggcctttgtcagaactagaatttgattattaacataggtgaaagttaat2220 ccactgtgactttgcccattgtttagaaagaatattcatagtttaattatgccttttttg2280 atcaggcacagtggctcacgcctgtaatcctagcagtttgggaggctgagccgggtggat2340 cgcctgaggtcaggagttcaagacaagcctggcctacatggttgaaaccccatctctact2400 aaaaatacacaaattagctaggcatggtggactcgcctgtaatctcactacacaggaggc2460 tgaggcaggagaatcacttgaacctgggaggcggatgttgaagtgagctgagattgcacc252-0 actgcactccagtctgggtgagagtgagactcagtcttaaaaaaatatgcctttttgaag2580 cacgtacattttgtaacaaagaactgaagctcttattatattattagttttgatttaatg2640 ttttcagcccatctcctttcatatttctgggagacagaaaacatgtttccctacacctct2700 tgcattccatcctcaacacccaactgtctcgatgcaatgaacacttaataaaaaacagtc2760 gattggtc 2768 <210> 15 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 15 ccggaattca ggaaagacat gatactgtcg gcagaagcc 39 <210> 16 <211> 41 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 16 cgcggatccg gccgctgcag gcgcagaact ggtaggtatg g 41 <210> 17 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 17 gatgtgctcc aggctaaagt t 21 <210> 18 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 18 agaaacggaa tgttgtggag t 21 <210> 19 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 19 ccgcctctag agcggtttct taccaata 2g <210> 20 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 20 ccgccggatc cagccagagt aggcaaatct 30 <210> 21 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 21 ggatgaatat gccctacatg <210> 22 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 22 tgatgggcag caggtctcat 20 <210> 23 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 23 acagaaacta tgtgagcctg 20 <210> 24 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Primer <400> 24 atgggacggt tcacatgttc 20 <210> 25 <211> 1450 <212> DNA
<213> Human <400>

gtgagcttgctccttaagttacaggaactctccttataatagacacttcattttcctagt60 ccatccctcatgaaaaatgactgaccactgctgggcagcaggagggatgataatcctaac120 tccaatcactggcaactcctgagatcagag,gaaaaccagcaacagcgtgg.gagtttgggg180 agaggcattccataccagattctgtggcctgcaggtgacatgctgcctaagagaagcagg240 agtctgtgacagccaccccaacacgtgacgtcatggccagtagggaagatgagctgagga300 actgtgtggtatgtggggaccaagccacaggctaccactttaatgcgctgacttgtgagg360 gctgcaagggtttcttcaggagaacagtcagcaaaagcattggtcccacctgcccctttg420 ctggaagctgtgaagtcagcaagactcagagg,cgccactgcccagcctgcaggttgcaga480 agtgcttagatgctggcatgaggaaagacatgatactgtcggcagaagccctggcattgc540 ggcgagcaaagcaggcccagcggcgggcacagcaaacacctgtgcaactgagtaaggagc600 aagaagagctgatccggacactcctgggggcccacacccgccacatgggcaccatgtttg660 aacagtttgtgcagtttaggcctccagctcatctgttcatccatcaccagcccttgccca720 ccctggcccctgtgctgcctctggtcacacacttcgcagacatcaacactttcatggtac780 tgcaagtcatcaagtttactaaggacctgcccgtcttccgttccctgcccattgaagacc840 agatctcccttctcaagggagcagctgtggaaatctgtcacatcgtactcaataccactt900 tctgtctccaaacacaaaacttcctctgcgggcctcttcgctacacaattgaagatggag960 cccgtgtggggttccaggtagagtttttggagttgctctttcacttccatggaacactac1020 gaaaactgcagctccaagagcctgagtatgtgctcttggctgccatggccctcttctctc1080 ctgaccgacctggagttacccagagagatgagattgatcagctgcaagaggagatggcac1140 tgactctgcaaagctacatcaagggccagcagcgaaggccccgggatcggtttctgtatg1200 cgaagttgctaggcetgctggctgagctccggagcattaatgaggcctacgggtaccaaa1260 tccagcacatccagggcctgtctgccatgatgccgctgctccaggagatctgcagctgag1320 gccatgctcacttccttccccagctcacctggaacaccctggatacactggagtgggaaa1380 atgctgggac caaagattgg gccgggttca aagggagccc agtggttgca atgaaagact 1440 aaagcaaaac 1450 <210> 26 <211> 1350 <212> DNA
<213> Mou5e <400>

cttgttttccaggcactgaggaccgcagtccctaattcctggcagttcctgagatctcaa60 ggaaagcagggtcagcgaggaggcctggggagaggaggcatcctacacccaatcttgtgg120 cct'gctgcctaagggaaacaggag.accatgacagctatgctaacactagaaaccatggcc180 agtgaagaagaatatgggccgaggaactgtgtggtgtgtggaga.ccgggc.cacaggctat240 catttccacgccctgacttgtgagggctgcaagggcttcttcagacgaacagtcagcaaa300 accattggtcccatctgtccgtttgctggaaggtgtgaggtcagcaaggcccagagacgc360 cactgtccagcctgcaggttgcagaagtgtctaaatgttggcatgaggaaagacatgata420 ctgtcagcagaagccctggcattgcggcgagccagacaggcacagcggcgggcagagaaa 480 gcatctttgcaactgaatcagcagcagaaagaactggtccagatcctcctgggggcccac 540 actcgccatgtgggcccattgtttgaccagtttgtgcagttcaagcctccagcctatctg 600 ttcatgcatcaccggcctttccagcctcggggccccgtgttgcctctgctcacacacttt 660 gcagatatcaacacgtttatggtgcaacagatcatcaagttcaccaaggatctgccgctc 720 ttccggtccctaaccatggaggaccagatctcccttctcaagggagcggctgtggaaata780 ttgcatatctcactcaacactacgttctgtcttcaaacagagaatttcttctgtgggcct840 ctttgctacaagatggaggacgcagtccatgcagggttccagtacgagtttttggagtcg900 atcctccacttccataaaaacctgaaaggactgcatctccaggagcctgagtatgtgctc960 atggctgccacggccctcttctcccctgacagacccggagttacccaaagagaagagata1020 gatcagctac aagaggagat ggcgctgatt ctgaacaacc acattatgg.a acaacagtct 1080 cggctccaaa gtcggtttct gtatgcaaag ctgatgggcc tgctggctga cctccggagt 1140 ataaacaatg catactccta tgaacttcag cgcttggagg aactgtctgc tatgacgccg 1200 ctgctcgggg agatttgcag ttgaggccca ggCttgCatC CtttCCCCag acccccaggg 1260 atacactggc ctggaaaggg tacagcgctg gaccccacac gggaaccagc aggaaggagc 1320 ttgggagtgg caatgaaatg ctgaacagtc 1350 <210> 27 <211> 286 <212> PRT
<213> Mouse <400> 27 Met Thr Ala Met Leu Thr Leu Glu Thr Met Ala Ser Glu GTu Glu Tyr 1 5 10 l5 Gly Pro Arg Asn Cys Val Val Cys Gly Asp Arg Ala Thr Gly Tyr His Phe His Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Val Ser Lys Thr Ile Gly Pro Ile Cys Pro Phe Ala Gly Arg Cys Glu Val Ser Lys Ala Gln Arg Arg His Cys Pro Ala Cys Arg Leu Gln Lys Cys Leu Asn Val Gly Met Arg Lys Asp Met Ile Leu Ser Ala Glu Ala Leu Ala Leu Arg Arg Ala Arg Gln Ala Gln Arg Arg Ala Glu Lys Ala Ser Leu Gln Leu Asn G1n Gln Gln Lys Glu Leu Val Gln Ile Leu Leu Gly Ala His Thr Arg His Val Gly Pro Leu Phe Asp Gln Pile Val Gln Phe Lys Pro Pro Ala Tyr Leu Phe Met His His Arg Pro Phe Gln Pro Arg Gly Pro Val Leu Pro Leu Leu Thr His Phe Ala Asp Ile Asn Thr 165 l70 175 Phe Met Val Gln Gln Ile Ile Lys Phe Thr Lys Asp Leu Pro Leu Phe Arg Ser Leu Thr Met Glu Asp Gln Ile Ser Leu Leu Lys Gly Ala Ala Val Glu Ile Leu His Ile Ser Leu Asn Thr Thr Phe Cys Leu Gln Thr Glu Asn Phe Phe Cys Gly Pro Leu Cys Tyr Lys Met Glu Asp Ala Val His Ala Gly Phe Gln Tyr Glu Phe Leu Glu Ser Ile Leu His Phe His Lys Asn Leu Lys Gly Leu His Leu Gln Glu Pro Glu Tyr Val Leu Met Ala Ala Thr Ala Leu Phe Ser Pro Gly Phe Cys Met Gln Ser

Claims (65)

Claims

1. A transgenic mouse expressing a human CAR receptor.

2. The transgenic mouse of claim 1, wherein said mouse does not express a murine CAR receptor having at least 60% of the naturally occurring murine CAR
receptor activity.

3. The transgenic mouse of claim 1, wherein said mouse does not express a murine CAR receptor.

4. The transgenic mouse of claim 1, wherein said human CAR receptor induces the expression of a CAR target gene.

5. The transgenic mouse of claim 4, wherein said CAR target gene is CYP2B 10 or CYP2B6.

6. The transgenic mouse of claim 5, wherein said CAR target gene is CYP2B 10.

7. The transgenic mouse of claim 4, wherein said CAR target gene is CYP3A 11 or CYP3A4.

8. The transgenic mouse of claim 4, wherein said human CAR receptor is activated by phenobarbital.

9. The transgenic mouse of claim 4, wherein activity of said human CAR
receptor is increased by less than 50% by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene.

10. The transgenic mouse of claim 4, wherein phenobarbital increases an activity of said human CAR receptor by at least 2 fold more than the corresponding amount of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene increases said activity of said human CAR
receptor.

11. The transgenic mouse of claim 4, wherein said human CAR receptor is activated by acetaminophen.

12. The transgenic mouse of claim 1, wherein the mRNA of said human GAR
receptor comprises a bovine growth hormone polyadenylation sequence.

13. The transgenic mouse of claim 1, wherein the nucleic acid encoding said human CAR receptor is operably linked to an intronic sequence of a rabbit .beta.-globin nucleic acid.

14. The transgenic mouse of claim 13, wherein said intronic sequence increases the expression of said human CAR receptor.

15. A transgenic mouse expressing a human CAR receptor, wherein said transgenic mouse exhibits an induction of a xenobiotic response in the presence of a xenobiotic inducer.

16. The transgenic mouse of claim 15, wherein said xenobiotic response comprises an induction or activation of a CAR target gene.

17. The mouse of claim 16, wherein said CAR target gene is CYP2B10.

18. The transgenic mouse of claim 15, wherein said xenobiotic inducer is phenobarbitol.

19. The transgenic mouse of claim 15, wherein said transgenic mouse does not express murine CAR.

20. The transgenic mouse of claim 15, wherein said human CAR receptor is the human CAR ligand binding domain.

21. A transgenic mouse comprising a mutation in a CAR gene, wherein, said comprises a reduction or absence of CAR receptor activity.

22. The transgenic mouse of claim 21, wherein said transgenic mouse exhibits a decrease or absence in induction, expression, or activity of a CAR target genes in response to a xenobiotic inducer.

23. The transgenic mouse of claim 21, wherein said xenobiotic inducer is phenobarbitol or 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene.

24. The transgenic mouse of claim 21, wherein said transgenic mouse does not exhibit hyperplasia or hypertrophy of the liver upon treatment with an xenobiotic inducer.

25. The transgenic mouse of claim 24, wherein said xenobiotic inducer is phenobarbitol or 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene.

26. The transgenic mouse of claim 21, wherein said transgenic mouse exhibits decreased metabolism of zoxazolamine.

27. The transgenic mouse of claim 21, wherein said transgenic mouse exhibits a resistance to acetaminophen toxicity.

28. A cell or tissue isolated from the transgenic mouse of claim 15 or claim 21.

29. A screening method for determining whether a compound activates a CAR
receptor, said method comprising the steps of:
(a) administering a compound to a transgenic mouse expressing a human CAR
receptor; and (b) measuring induction of a CAR target gene, whereby said compound is determined to activate said CAR receptor if said compound mediates induction of said CAR target gene.

30. The method of claim 29, wherein step (a) further comprises administering a CAR receptor inverse agonist to said mouse expressing said human CAR receptor.

31. The method of claim 30, wherein said CAR receptor inverse agonist is clotrimazole.

32. A screening method for determining whether a compound inhibits a CAR
receptor, said method comprising the steps of:
(a) administering said compound to a transgenic mouse expressing a human CAR
receptor; and (b) measuring expression of a CAR target gene in the presence and absence of said compound, whereby said compound is determined to inhibit said CAR
receptor if said compound decreases said expression of said CAR target gene.

33. The method of claim 32, wherein step (a) further comprises administering a CAR receptor agonist to said mouse expressing said human CAR receptor.

34. The screening method of claim 33, wherein said CAR receptor agonist is administered after said compound.

35. A screening method for determining whether a compound modulates the activity of a CAR receptor, said method comprising the steps of:
(a) administering said compound to a transgenic mouse expressing a human CAR
receptor; and (b) measuring a physiological effect mediated by the administration of said compound, whereby said compound is determined to modulate the activity of said CAR
receptor if the magnitude of said physiological effect in said mouse expressing said human receptor differs from that in a mouse comprising a mutation that reduces CAR
receptor activity.

36. The method of claim 35, wherein said measuring said physiological effect comprises measuring the toxicity or activity mediated by the administration of said compound or measuring the half-life of said compound.

37. The method of claim 36, wherein said toxicity or activity is mediated by a metabolite of said compound.

38. A screening method for determining whether the metabolism of a compound is regulated by modulation of the activity of a CAR receptor, said method comprising the steps of:
(a) administering said compound to a transgenic mouse expressing a human CAR
receptor; and (b) measuring the rate of metabolism of said compound, whereby said metabolism of said compound is determined to be regulated by modulation of the activity of said CAR receptor if said rate of metabolism is faster in said mouse expressing said human receptor than in a mouse comprising a mutation that reduces CAR receptor activity.

39. The method of claim 38, wherein said measuring said rate of metabolism comprises measuring the toxicity or activity mediated by the administration of said compound, measuring the half-life of said compound, or measuring the serum level of a liver enzyme.

40. A screening method for determining whether the metabolism of a first compound is modulated by a second compound, said method comprising the steps of:
(a) administering said first compound in the presence and absence of said second compound to a transgenic mouse expressing a human CAR receptor; and (b) in the presence and absence of said second compound, measuring a physiological effect that is mediated by the administration of said first compound, whereby said second compound is determined to modulate the metabolism of said first compound if said second compound effects a change in said physiological effect mediated by said administration of said first compound.

41. The method of claim 40, wherein said measuring said physiological effect comprises measuring the toxicity or activity mediated by the administration of said first compound or measuring the half-life of said first compound.

42. The method of claim 41, wherein said toxicity or activity is mediated by a metabolite of said first compound.

43. The method of claim 41, wherein step (b) comprises measuring the half-life of said first compound in the presence and absence of said second compound, whereby said second compound is determined to activate the metabolism of said first compound if said second compound decreases said half-life, or said second compound is determined to inhibit the metabolism of said first compound if the said second compound increases said half-life.

44. The method of claim 29, 32, 35, 38, or 40, wherein said mouse expressing said human CAR receptor does not express a substantially active murine CAR
receptor.

45. A screening method for determining whether a compound activates a CAR
receptor, said method comprising the steps of:
(a) administering a compound to a mouse, said mouse comprising a mutation that reduces CAR receptor activity; and (b) measuring induction of a CAR target gene, whereby said compound is determined to activate said CAR receptor if said induction is smaller in said mouse comprising said mutation than in a mouse having wild-type CAR receptor activity.

46. The method of claim 45, wherein step (a) further comprises administering a CAR receptor inverse agonist to said mouse comprising said mutation.

47. The method of claim 46, wherein said CAR receptor inverse agonist is androstanol.

48. A screening method for determining whether a compound inhibits a CAR
receptor, said method comprising the steps of:
(a) administering said compound to a mouse, said mouse comprising a mutation that reduces CAR receptor activity; and (b) measuring expression of a CAR target gene in the presence and absence of said compound, whereby said compound is determined to inhibit said CAR
receptor if the decrease in said expression effected by said compound is smaller in said mouse comprising said mutation than in a mouse having wild-type CAR receptor activity.

49. The method of claim 48, wherein step (a) further comprises administering a CAR receptor agonist to said mouse comprising said mutation.

50. The method of claim 49, wherein said CAR receptor agonist is TCPOBOP, and said TCPOBOP is administered after said compound.

51. A screening method for determining whether a compound modulates the activity of a CAR receptor, said method comprising the steps of:
(a) administering said compound to a mouse, said mouse comprising a mutation that reduces CAR receptor activity; and (b) measuring a physiological effect mediated by the administration of said compound, whereby said compound is determined to modulate the activity of said CAR
receptor if the magnitude of said physiological effect in said mouse comprising said mutation differs from that in a mouse having wild-type CAR receptor activity.

52. The method of claim 51, wherein said measuring said physiological effect comprises measuring the toxicity or activity mediated by the administration of said compound or measuring the half-life of said compound.

53. The method of claim 52, wherein said toxicity or activity is mediated by a metabolite of said compound.

54. A screening method for determining whether the metabolism of a compound is regulated by modulation of the activity of a CAR receptor, said method comprising the steps of:
(a) administering said compound to a mouse, said mouse comprising a mutation that reduces CAR receptor activity; and (b) measuring the rate of metabolism of said compound, whereby said metabolism of said compound is determined to be regulated by modulation of the activity of said CAR receptor if said rate of metabolism is slower in said mouse comprising said mutation than in a mouse having wild-type CAR receptor activity.

55. The method of claim 54, wherein said measuring said rate of metabolism comprises measuring the toxicity or activity mediated by the administration of said compound, measuring the half-life of said compound, or measuring the serum level of a liver enzyme.

56. A screening method for determining whether the metabolism of a first compound is modulated by a second compound, said method comprising the steps of:
(a) administering said first compound in the presence and absence of said second compound to a mouse, said mouse comprising a mutation that reduces CAR
receptor activity; and (b) in the presence and absence of said second compound, measuring a physiological effect that is mediated by the administration of said first compound, whereby said second compound is determined to modulate the metabolism of said first compound if the change effected by said second compound in said physiological effect mediated by said administration of said first compound is smaller in said mouse comprising said mutation than in a mouse having wild-type CAR receptor activity.

57. The method of claim 56, wherein said measuring said physiological effect comprises measuring the toxicity or activity mediated by the administration of said first compound or measuring the half-life of said first compound.

58. The method of claim 57, wherein said toxicity or activity is mediated by a metabolite of said first compound.

59. The method of claim 57, wherein step (b) comprises measuring the half-life of said first compound in the presence and absence of said second compound, whereby said second compound is determined to activate the metabolism of said first compound if the decrease in said half-life effected by said second compound is smaller in said mouse comprising said mutation than in a mouse having wild-type CAR receptor activity, or said second compound is determined to inhibit the metabolism of said first compound if the increase in said half-life effected by said second compound is smaller in said mouse comprising said mutation than in a mouse having wild-type CAR receptor activity.

60. The method of claim 45, 48, 51, 54, or 56, wherein said mouse is a transgenic mouse.

61. The method of claim 45, 48, 51, 54, or 56, wherein said mutation substantially eliminates CAR receptor activity.

62. The method of claim 29, 38, 45, or 54, wherein said compound is eliminated from drug development.

63. The method of claim 40 or 56, wherein said first compound activates the metabolism of said second compound, and said first compound or said second compound is eliminated from drug development.

64. The method of claim 29, 32, 45, or 48 wherein said CAR target gene is CYP2B10 or CYP2B6.

65. The method of claim 29, 32, 35, 38, 40, 45, 48, 51, 54, or 56, wherein at least one of said compound, said first compound, or said second compound is a member of a library of at least 5 compounds, all of which are simultaneously administered to said mouse comprising said mutation or said mouse expressing said human CAR
receptor.